Novel sigma receptor ligands and methods of modulating cellular protein homeostasis using same

ABSTRACT

The present invention includes compounds useful in preventing, treating or ameliorating Sigma-related disorders or diseases. The compounds of the invention may modulate cellular protein homeostasis, which includes: translation initiation, folding, processing, transport, and degradation (including ubiquitin selective autophagy) of proteins. The present invention also includes methods of preventing, treating or ameliorating a Sigma-related disorder or disease in a subject in need thereof, the method comprising administering to the subject an effective amount of a Sigma-modulating compound. The present invention also includes methods of preventing, treating or ameliorating a Sigma-related disorder or disease in a subject in need thereof, the method comprising administering to the subject an effective amount of a Sigma-modulating compound, further comprising administering an effective amount of a compound that inhibits the ubiquitin proteasome system (UPS) and/or autophagic survival pathways.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation application of, and claimspriority to, U.S. patent application Ser. No. 15/176,812, filed Jun. 8,2016, now issued as U.S. Pat. No. 9,889,102, which is a continuation ofU.S. patent application Ser. No. 14/415,061, filed Jan. 15, 2015, nowissued as U.S. Pat. No. 9,388,126, which is the U.S. national phaseapplication filed under 35 U.S.C. § 371 claiming priority to PCTInternational Application No. PCT/US2013/051110, filed Jul. 18, 2013,which claims priority under 35 U.S.C. § 119(e) to U.S. ProvisionalPatent Application No. 61/673,565, filed Jul. 19, 2012, all of whichapplications are hereby incorporated herein by reference in theirentireties.

BACKGROUND OF THE INVENTION

In eukaryotic cells the endoplasmic reticulum (ER) is the primary siteof synthesis, folding, and assembly of secreted and integral membraneproteins and their macromolecular complexes (Mu et al., 2008, Cell134:769-781; Marciniak et al., 2006, Physiol. Rev. 2006:1133-1149; Ronet al., 2007, Nat. Rev. Mol. Cell Biol. 519-529). Maintenance of ERprotein homeostasis relies on the timely convergence of multiplepathways that detect homeostatic protein concentration thresholds andcontrol the ebb-and-flow of ER proteins (Mu et al., 2008, Cell134:769-781; Marciniak et al, 2006, Physiol. Rev. 2006:1133-1149; Ron etal., 2007, Nat. Rev. Mol. Cell Biol. 519-529; Jonikas et al. 2009,Science 323:1693-1697). This process is driven by an intricate networkof molecular chaperones and transcription factors. Disruption of ERhomeostasis activates stress response pathways including the unfoldedprotein response (UPR) (Marciniak et al, 2006, Physiol. Rev.2006:1133-1149; Ron et al., 2007, Nat. Rev. Mol. Cell Biol. 519-529; Kimet al., 2008, Nat. Rev. Drug Discov. 7:1013-1030; Xu et al., 2005, J.Clin. Invest. 2656-2664).

The mammalian UPR comprises at least two phases: an initial alarm phase,followed by a cytoprotective, adaptive phase in which UPR factors areupregulated to enhance the cellular capacity to process increasedconcentrations of unfolded protein (Marciniak et al, 2006, Physiol. Rev.2006:1133-1149; Ron et al., 2007, Nat. Rev. Mol. Cell Biol. 519-529; Kimet al., 2008, Nat. Rev. Drug Discov. 7:1013-1030; Xu et al., 2005, J.Clin. Invest. 2656-2664). Imbalanced or altered capacity to respond toER stress has been implicated in various diseases and disorders(Marciniak et al, 2006, Physiol. Rev. 2006:1133-1149; Kim et al., 2008,Nat. Rev. Drug Discov. 7:1013-1030; Ma et al., 2004, Nat. Rev. Cancer4:966-977). Protracted ER stress can overwhelm the UPR, leading toautophagy as a secondary survival response (Ron et al., 2007, Nat. Rev.Mol. Cell Biol. 519-529; Bernales et al., 2006, PLoS Biol. 4:e423; Ogataet al., 2006, Mol. Cell Biol. 26:9220-9231; Yorimitsu et al., 2006, J.Biol. Chem. 281:30299-30304). Although the relationship between ERstress, unfolded protein response, and autophagy remains unclear,growing evidence suggests that these responses are likely integratedsignaling pathways that modulate cell survival and growth (Ron et al.,2007, Nat. Rev. Mol. Cell Biol. 519-529, He et al., 2009, Annu. Rev.Genet. 43:67-93, Hoyer-Hansen et al., 2007, Cell Death Differ.14:1576-1582).

Autophagy describes a set of bulk cellular degradation pathways in whichlarge aggregates of misfolded proteins and damaged cellular components,including damaged organelles, are sequestered into membrane boundvesicles called autophagosomes and subsequently targeted for lysosomaldegradation (He et al., 2009, Annu. Rev. Genet. 43:67-93; Levine et al.,2004, Dev. Cell 6:463-477). Complete autophagy comprises autophagosomefusion with lysosomes to form autolysosomes, wherein the sequesteredproteins and lipids are subsequently degraded by autophagic degradationor flux (He et al., 2009, Annu. Rev. Genet. 43:67-93; Levine et al.,2004, Dev. Cell 6:463-477). Autophagy occurs under basal conditions inmany tissues and is involved in cellular differentiation anddevelopment. It is also activated or hyperactivated in conditions ofnutrient starvation and cellular stress (Levine et al., 2004, Dev. Cell6:463-477, Mizushima et al., 2008, Nature 451:1069-1075), to maintainenergy levels and to sequester and remove damaged and cytotoxic cellularcomponents (Levine et al., 2004, Dev. Cell 6:463-477; Mizushima et al.,2008, Nature 451:1069-1075). Thus, autophagy plays important roles incellular homeostasis and disease prevention, and defective autophagy hasbeen implicated in neurodegenerative disease and cancer (Levine et al.,2008, Cell 132:27-42; Mizushima et al., 2008, Nature 451:1069-1075;White et al., 2009, Clin. Cancer Res. 15:5308-5316).

Autophagy has been shown to influence tumor cell growth andtumorigenesis (Levine et al., 2008, Cell 132:27-42; White et al., 2009,Clin. Cancer Res. 15:5308-5316; Degenhardt et al., 2006, Cancer Cell10:304-312; Mathew et al., 2007, Nat. Rev. Cancer 7:961-967). Autophagymay serve a cytoprotective role in cancer cells (Levine et al., 2008,Cell 132:27-42; Mizushima et al., 2008, Nature 451:1069-1075; White etal., 2009, Clin. Cancer Res. 15:5308-5316; Degenhardt et al., 2006,Cancer Cell 10:304-312). Several antineoplastic agents have been shownto induce autophagy (Rubinsztein et al., 2007, Rev. Drug Discov.6:304-312). However, in many cases it remains unclear whether cell deathoccurs by autophagy, whether cell death is associated with autophagy, orwhether autophagy is a survival response to cytotoxic chemotherapy(Levine et al., 2004, Dev. Cell 6:463-477; Levine et al., 2008, Cell132:27-42; White et al., 2009, Clin. Cancer Res. 15:5308-5316; Hippertet al., 2006, Cancer Res. 66:9349-9351). Emerging data suggest thatautophagy participates in integrated responses to cellular stress thatdetermine cell death versus survival. The proteins and pathways thatregulate these integrated stress responses are just beginning to bedefined (Ron et al., 2007, Nat. Rev. Mol. Cell Biol. 519-529; Kim etal., 2008, Nat. Rev. Drug Discov. 7:1013-1030, Levine et al., 2004, Dev.Cell 6:463-477; Rubinsztein et al., 2006, Neuron 54:9349-9351).

Sigma receptors, first proposed 30 years ago (Martin et al., 1976, J.Pharmacol. Exp. Ther. 197:517-532), are distinct from classical opioidreceptors (Su, 1982, J. Pharmacol. Exp. Ther. 223:284-290). Bindingstudies suggest at least two Sigma receptor subtypes, of which only theSigma1 receptor (hereinafter “Sigma1”) has been cloned, whereas theidentity of Sigma2 remains unclear (Hanner et al., 1996, Proc. Natl.Acad. Sci. U.S.A. 93:8072-8077; Vilner et al., 1995, Cancer Res.55:408-413). Sigma1 is highly conserved among mammals (greater than 80%amino acid identity), but shares no significant homology with anytraditional receptor family or other mammalian protein (White et al.,2009, Clin. Cancer Res. 15:5308-5316; Mathew, et al., 2007, Nat. Rev.Cancer 7:961-967). Cloned Sigma1 is a 26 kilodalton integral membraneprotein (Hanner et al., 1996, Proc. Natl. Acad. Sci. U.S.A.93:8072-8077; Pal et al., 2007, Mol. Pharmacol. 72:921-933; Aydar etal., 2007, Neuron 34:399-410; Hayashi et al., 2007, Cell 131:596-610).It is found primarily in the ER, and can translocate to the plasmamembrane, other organelles, and endoplasmic membrane microdomains(Hanner et al., 1996, Proc. Natl. Acad. Sci. U.S.A. 93:8072-8077; Aydaret al., 2007, Neuron 34:399-410; Hayashi et al., 2007, Cell 131:596-610;Hayashi et al., 2003, J. Pharmacol. Exp. Ther. 306:718-725; Palmer etal., 2007, Cancer Res. 67:11166-11175). Sigma receptors are highlyexpressed in tumor cell lines, including prostate and breastadenocarcinoma (Vilner et al., 1995, Cancer Res. 55:408-413; Berthosiset al., 2003, Br. J. Cancer 88:438-446; Piergentili et al., J. Med.Chem. 53:1261-1269). Some Sigma ligands are reported as antitumor agents(Berthosis et al., 2003, Br. J. Cancer 88:438-446; Vilner et al., 1995,J. Neurosci. 15:117-134). Interestingly, putative Sigma antagonists, butnot agonists, inhibit prostate carcinoma proliferation in vitro andinhibit tumor growth in tumor xenograft experiments (Berthosis et al.,2003, Br. J. Cancer 88:438-446; Spruce et al., 2004, Cancer Res.64:4875-4886). Recent work has described Sigma ligand-induced cell deathby lysosomal destabilization and oxidative stress.

There are numerous examples of clinically used compounds that bindSigma1 with high affinity and thus are considered Sigma1 ligands, suchas haloperidol, a widely used antipsychotic that also binds D2 receptorswith similar affinity and whose anti-psychotic properties are primarilyunderstood as D2 mediated (Seeman, et al., 1975, Science 188:1217-1219;Seeman et al., 1976, Nature 261:717-719), and the hallucinogenN,N-dimethyltryptamine, hypothesized to be an endogenous Sigma1regulator (Fontanilla et al., 2009, Science 323:934-937). Sigmareceptors have proved to be highly attractive pharmacological targetsfor the treatment of various pathologies, such as neuropathic pain (dela Puente et al., 2009, Pain 145:294-303), depression (Skuza, 2003, Pol.J. Pharmacol. 55:923-934), cocaine abuse (Matsumoto et al., 2003, Eur.J. Pharmacol. 469:1-12), epilepsy (Lin et al., 1997, Med. Res. Rev.17:537-572), psychosis (Rowley et al., 2001, J. Med. Chem. 44:477-501),and Alzheimer's and Parkinson's disease (Maurice et al., 1997, Prog.Neuro-Psychopharmacol. Biol. Psychiatry 21:69-102; Marrazzo et al.,2005, NeuroReport 16:1223-1226). Recent reports demonstrate a geneticlink between the Sigma1 receptor gene (SIGMAR1) and Amyotrophic lateralsclerosis (ALS) (Al-Saif et al., 2011, Ann Neurol. 70(6):913-9), as wellas Frontotemporal Lobar Degeneration (FTLD) (Luty et al., 2010, AnnNeurol. 2010 68(5):639-49). Moreover, Sigma1 antagonists and Sigma2agonists may be useful as anticancer agents and selective tumor imagingagents (Akhter et al., 2008, Nucl. Med. Biol. 35:29-34; Tu et al., 2007,J. Med. Chem. 50:3194-3204).

Sigma1 can function as a molecular chaperone at the ER-mitochondrioninterface at least in certain model cell lines (Hayashi & Su, 2007, Cell131(3):596-610). However, the physiological role of Sigma receptors aswell as their role in neurodegenerative disease and cancer remainsunclear. In vitro, treatment with a Sigma antagonist results inapoptotic cell death following prolonged treatment, with Sigma ligandtime-action and dose-response, depending on the Sigma antagonist andcell line (Berthosis et al., 2003, Br. J. Cancer 88:438-446; Piergentiliet al., J. Med. Chem. 53:1261-1269; Spruce et al., 2004, Cancer Res.64:4875-4886; Vilner et al., 1995, J. Neurosci. 15:117-134). Yet, amechanistic understanding of the Sigma1 receptor system remains elusive.

Most prostate cancer patients become unresponsive to initially effectivehormone- and chemotherapy as prostate tumor cells eventually adapt anddevelop resistance. Treatment with Sigma antagonists leads to apoptoticcell death of both androgen-sensitive and androgen-insensitive prostatecancer cells (Berthosis et al., 2003, Br. J. Cancer 88:438-446; Spruceet al., 2004, Cancer Res. 64:4875-4886). Although some insight has beengained into how prostate cancer cells develop such resistance, currentlythere are few alternatives to treat hormone refractory (castrationresistant) prostate cancer. Emerging therapies to treat intractable,advanced prostate cancers target protein processing and chaperonepathways that maintain prostate tumor growth and survival.

There is a need in the art to identify compounds useful in the treatmentof intractable, advanced cancers. Such compounds may target proteinprocessing, protein synthesis, protein folding, protein transport,protein localization, protein assembly into functional macromolecularcomplexes, and related chaperone pathways, all of which may helpmaintain tumor growth, survival and metastasis. The present inventionaddresses this unmet need.

BRIEF SUMMARY OF THE INVENTION

The present invention includes a composition comprising at least onecompound selected from the group consisting of:

(i) a compound of Formula (I):

wherein:

ring A is a monocyclic or bicyclic aryl or a monocyclic or bicyclicheteroaryl ring, and wherein the aryl or heteroaryl ring is optionallysubstituted with 0-4 R¹ groups;

each occurrence of R¹ is independently selected from the groupconsisting of —C₁-C₆ alkyl, —C₁-C₆ fluoroalkyl, —C₁-C₆ heteroalkyl, F,Cl, Br, I, —CN, —NO₂, —OR³, —SR³, —S(═O)R³, —S(═O)₂R³, —NHS(═O)₂R³,—C(═O)R³, —OC(═O)R³, —CO₂R³, —OCO₂R³, —CH(R³)₂, —N(R³)₂, —C(═O)N(R³)₂,—OC(═O)N(R³)₂, —NHC(═O)NH(R³), —NHC(═O)R³, —NHC(═O)OR³, —C(OH)(R³)₂, and—C(NH₂)(R³)₂;

each occurrence of R² is independently selected from the groupconsisting of H, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, and —C₁-C₃ alkyl-(C₃-C₆cycloalkyl), wherein the alkyl, heteroalkyl or cycloalkyl group isoptionally substituted with 0-5 R¹ groups, or X³ and R² combine to forma (C₃-C₇)heterocycloalkyl group, optionally substituted with 0-2 R¹groups;

each occurrence of R³ is independently selected from the groupconsisting of H, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, aryl, and —C₁-C₃alkyl-(C₃-C₆ cycloalkyl), wherein the alkyl, heteroalkyl, aryl, orcycloalkyl group is optionally substituted with 0-5 R¹ groups;

X¹ is —CH₂—, —S—, —O— or —(NR²)—;

X² is ═CH₂, ═S, ═O or ═NR²; and

X³ is —S—, —O—, or —NR²—; and

(ii) a compound of Formula (II):

R^(A)—R^(B)  (II), wherein;

R^(A) is selected from the group consisting of

X⁴ is selected from the group consisting of F, Cl, Br, and I; and

R^(B) is selected from the group consisting of:

(iii) a salt, solvate, or N-oxide thereof, andany combinations thereof.

In one embodiment, in Formula (I) ring A is a monocyclic aryl ormonocyclic heteroaryl ring optionally substituted with 0-4 R¹ groups. Inanother embodiment, in Formula (I) ring A is phenyl optionallysubstituted with 0-4 R¹ groups. In yet another embodiment, in Formula(I) X¹ and X³ are both —NH—, and X² is ═NH.

In yet another embodiment, the compound of Formula (I) is selected fromthe group consisting of1-(3-(4-fluorophenoxy)propyl)-3-(4-iodophenyl)guanidine (Compound A),1-(3-(4-fluorophenoxy)propyl)-3-(4-methoxyphenyl)guanidine (Compound B),1-(n-propyl)-3-(4-iodophenyl)guanidine (Compound C),1-(n-propyl)-3-(4-methoxyphenyl)guanidine (Compound D),1-(3-(4-fluorophenoxy)propyl)-3-(4-trifluoromethylphenyl)guanidine(Compound F), 1-(3-(4-fluorophenoxy)propyl)-3-(4-chlorophenyl)guanidine(Compound G), a salt, solvate or N-oxide thereof, and any combinationsthereof.

In yet another embodiment, the compound of Formula (II) is selected fromthe group consisting of, 1,3-bis(3-(4-fluorophenoxy)propyl)guanidine(Compound E),1-(3-(4-fluorophenoxy)propyl)-3-(4-methyl-2-oxo-2H-chromen-7-yl)guanidine)(Compound H), a salt, solvate or N-oxide thereof, and any combinationsthereof.

The present invention also includes a composition comprising at leastone compound of Formula (III):

wherein within Formula (III);

each occurrence of R¹ and R² is independently selected from the groupconsisting of —C₁-C₆ alkyl, —C₁-C₆ fluoroalkyl, —C₁-C₆ heteroalkyl, F,Cl, Br, I, —CN, —NO₂, —OR⁵, —SR⁵, —S(═O)R⁵, —S(═O)₂R⁵, —NHS(═O)₂R⁵,—C(═O)R⁵, —OC(═O)R⁵, —CO₂R⁵, —OCO₂R⁵, —CH(R⁵)₂, —N(R⁵)₂, —C(═O)N(R⁵)₂,—OC(═O)N(R⁵)₂, —NHC(═O)NH(R⁵), —NHC(═O)R⁵, —NHC(═O)OR⁵, —C(OH)(R⁵)₂, and—C(NH₂)(R⁵)₂;

R³ is selected from the group consisting of —C₁-C₆ alkyl, —C₁-C₆fluoroalkyl, —C₁-C₆ alkoxy, F, Cl, Br, and I;

R⁴ is selected from the group consisting of —C₁-C₆ alkyl, —C₁-C₆ alkoxy,F, Cl, Br, and I;

each occurrence of R⁵ is independently selected from the groupconsisting of H, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, aryl, and —C₁-C₃alkyl-(C₃-C₆ cycloalkyl), wherein the alkyl, heteroalkyl, aryl, orcycloalkyl group is optionally substituted;

X is selected from the group consisting of CH₂, C═O, or O;

n is an integer from 1-3;

x is an integer from 0-4; and

y is an integer from 0-4;

a salt, solvate, or N-oxide thereof, and any combinations thereof.

In one embodiment, the compound of Formula (III) is selected from thegroup consisting of1-(3-(4-fluorophenoxy)propyl)-3-(4-iodophenyl)guanidine (Compound A),1-(3-(4-fluorophenoxy)propyl)-3-(4-methoxyphenyl)guanidine (Compound B),1-(3-(4-fluorophenoxy)propyl)-3-(4-trifluoromethylphenyl)guanidine(Compound F), 1-(3-(4-fluorophenoxy)propyl)-3-(4-chlorophenyl)guanidine(Compound G), a salt, solvate or N-oxide thereof, and any combinationsthereof.

The compositions of the present invention may include certainembodiments. In one embodiment, the composition further comprises apharmaceutically acceptable carrier. In another embodiment, thecomposition further comprises at least one additional therapeutic agentthat inhibits the ubiquitin proteasome system (UPS) or autophagicsurvival pathway. In yet another embodiment, the therapeutic agent isselected from the group consisting of growth factor receptor inhibitors,monoclonal antibodies against growth factor receptors, hormone receptorantagonists, autophagy modulators, ER stress response inhibitors,proteasome inhibitors, p97/VCP inhibitors and any combinations thereof.

In yet another embodiment, the therapeutic agent is selected from thegroup consisting of octapeptide, somatostatin, analoguem, lanreotide,angiopeptin, dermopeptin, octreotide, pegvisomant, 3-methyladenine,chloroquine, hydroxychloroquine, wortmannin, eeyarestatin I, salubrinal,versipelostatin, 2H-isoindole-2-carboxylic acid, 4-fluoro-1,3-dihydro-,(2R,6S,12Z,13aS,14aR,16aS)-14a-[[(cyclopropylsulfonyl)amino]carbonyl]-6-[[(1,1-dimethylethoxy)carbonyl]amino]-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a-hexadecahydro-5,16-dioxocyclopropa[e]pyrrolo[1,2-a][1,4]diazacyclopentadecin-2-ylester (Danoprevir),adamantane-acetyl-(6-aminohexanoyl)₃-(leucinyl)₃-vinyl-(methyl)-sulfone,N-acetyl-L-leucyl-L-leucyl-L-methional,N-[(phenylmethoxy)carbonyl]-L-leucyl-N-[(1S)-1-formyl-3-methylbutyl]-L-leucinamide,(2R,3S,4R)-3-hydroxy-2-[(1S)-1-hydroxy-2-methylpropyl]-4-methyl-5-oxo-2-pyrrolidinecarboxy-N-acetyl-L-cysteinethioester, N—[N—N-acetyl-L-leucyl)-L-leucyl]-L-norleucine, lactacystin,4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride,(S)-1-carboxy-2-phenyl]-carbamoyl-arg-val-arginal, bovine pancreatictrypsin inhibitor, [(2S,2R)-3-amino-2-hydroxy-4-phenylbutanoyl]-L-leucine,N—[(S)-1-carboxy-isopentyl)-carbamoyl-alpha-(2-iminohexahydro-4-(S)-pyrimidyl]-L-glycyl-L-phenylalaninal,ethylenediamine-tetraacetic acid disodium salt dehydrate,acetyl-leucyl-leucyl-arginal, isovaleryl-val-val-AHMHA-ala-AHMHA whereAHMHA=(3S, 4S)-4-amino-3-hydroxy-6-methylheptanoic acid,N-alpha-L-rhamnopyranosyloxy (hydroxyphosphinyl)-L-leucyl-L-tryptophan,phenylmethanesulfonyl fluoride, bortezomib, carfilzomib, ONX 0912,NPI-0052, CEP-18770, MLN9708, disulfiram, epigallocatechin-3-gallate,salinosporamide A, PI3K inhibitors, lapatinib, rapamycin, rapalogs, HSPinhibitors, androgen receptor inhibitors, conjugation products of Sigmaligands with targeting components, a salt thereof, and any combinationsthereof.

The present invention also includes a method of preventing, treating orameliorating a Sigma receptor-related disorder or disease in a subjectin need thereof. The method comprises administering to the subject aneffective amount of a therapeutic composition comprising at least onecompound selected from the group consisting of:

(i) a compound of Formula (I):

wherein:

ring A is a monocyclic or bicyclic aryl or a monocyclic or bicyclicheteroaryl ring, and wherein the aryl or heteroaryl ring is optionallysubstituted with 0-4 R¹ groups;

each occurrence of R¹ is independently selected from the groupconsisting of —C₁-C₆ alkyl, —C₁-C₆ fluoroalkyl, —C₁-C₆ heteroalkyl, F,Cl, Br, I, —CN, —NO₂, —OR³, —SR³, —S(═O)R³, —S(═O)₂R³, —NHS(═O)₂R³,—C(═O)R³, —OC(═O)R³, —CO₂R³, —OCO₂R³, —CH(R³)₂, —N(R³)₂, —C(═O)N(R³)₂,—OC(═O)N(R³)₂, —NHC(═O)NH(R³), —NHC(═O)R³, —NHC(═O)OR³, —C(OH)(R³)₂, and—C(NH₂)(R³)₂;

each occurrence of R² is independently selected from the groupconsisting of H, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, and —C₁-C₃ alkyl-(C₃-C₆cycloalkyl), wherein the alkyl, heteroalkyl or cycloalkyl group isoptionally substituted with 0-5 R¹ groups, or X³ and R² combine to forma (C₃-C₇)heterocycloalkyl group, optionally substituted with 0-2 R¹groups;

each occurrence of R³ is independently selected from the groupconsisting of H, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, aryl, and —C₁-C₃alkyl-(C₃-C₆ cycloalkyl), wherein the alkyl, heteroalkyl, aryl, orcycloalkyl group is optionally substituted with 0-5 R¹ groups;

X¹ is —CH₂—, —S—, —O— or —(NR²)—;

X² is ═CH₂, ═S, ═O or ═NR²; and

X³ is —S—, —O—, or —NR²—; and

(ii) a compound of Formula (II):

R^(A)—R^(B)  (II), wherein;

R^(A) is selected from the group consisting of

X⁴ is selected from the group consisting of F, Cl, Br, and I; and

R^(B) is selected from the group consisting of:

(iii) haloperidol, IPAG, PB28, rimcazole, BD1063, BD1047, PREO84, NE100,(+)-SKF10047, (+)-pentazocine,(iv) a salt, solvate, or N-oxide thereof, andany combinations thereof.

In one embodiment, the compound of Formula (I) is selected from thegroup consisting of1-(3-(4-fluorophenoxy)propyl)-3-(4-iodophenyl)guanidine (Compound A),1-(3-(4-fluorophenoxy)propyl)-3-(4-methoxyphenyl)guanidine (Compound B),1-(n-propyl)-3-(4-iodophenyl)guanidine (Compound C),1-(n-propyl)-3-(4-methoxyphenyl)guanidine (Compound D),1-(3-(4-fluorophenoxy)propyl)-3-(4-trifluoromethylphenyl)guanidine(Compound F), 1-(3-(4-fluorophenoxy)propyl)-3-(4-chlorophenyl)guanidine(Compound G), a salt, solvate or N-oxide thereof, and any combinationsthereof.

In one embodiment, the compound of Formula (II) is selected from thegroup consisting of, 1,3-bis(3-(4-fluorophenoxy)propyl)guanidine(Compound E),1-(3-(4-fluorophenoxy)propyl)-3-(4-methyl-2-oxo-2H-chromen-7-yl)guanidine)(Compound H), a salt, solvate or N-oxide thereof, and any combinationsthereof.

The present invention also includes a method of preventing, treating orameliorating a Sigma receptor-related disorder or disease in a subjectin need thereof. The method comprises administering to the subject aneffective amount of a therapeutic composition comprising at least onecompound selected from the group consisting of:

(i) a compound of Formula (III):

wherein within Formula (III);

each occurrence of R¹ and R² is independently selected from the groupconsisting of —C₁-C₆ alkyl, —C₁-C₆ fluoroalkyl, —C₁-C₆ heteroalkyl, F,Cl, Br, I, —CN, —NO₂, —OR⁵, —SR⁵, —S(═O)R⁵, —S(═O)₂R⁵, —NHS(═O)₂R⁵,—C(═O)R⁵, —OC(═O)R⁵, —CO₂R⁵, —OCO₂R⁵, —CH(R)₂, —N(R⁵)₂, —C(═O)N(R⁵)₂,—OC(═O)N(R⁵)₂, —NHC(═O)NH(R⁵), —NHC(═O)R⁵, —NHC(═O)OR⁵, —C(OH)(R⁵)₂, and—C(NH₂)(R⁵)₂;

R³ is selected from the group consisting of —C₁-C₆ alkyl, —C₁-C₆fluoroalkyl, —C₁-C₆ alkoxy, F, Cl, Br, and I;

R⁴ is selected from the group consisting of —C₁-C₆ alkyl, —C₁-C₆ alkoxy,F, Cl, Br, and I;

each occurrence of R⁵ is independently selected from the groupconsisting of H, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, aryl, and —C₁-C₃alkyl-(C₃-C₆ cycloalkyl), wherein the alkyl, heteroalkyl, aryl, orcycloalkyl group is optionally substituted;

X is selected from the group consisting of CH₂, C═O, or O;

n is an integer from 1-3;

x is an integer from 0-4; and

y is an integer from 0-4;

(ii) haloperidol, IPAG, PB28, rimcazole, BD1063, BD1047, PRE084, NE100,(+)-SKF10047, (+)-pentazocine;(iii) a salt, solvate, or N-oxide thereof; andany combinations thereof.

In one embodiment, the compound of Formula (III) is selected from thegroup consisting of1-(3-(4-fluorophenoxy)propyl)-3-(4-iodophenyl)guanidine (Compound A),1-(3-(4-fluorophenoxy)propyl)-3-(4-methoxyphenyl)guanidine (Compound B),1-(3-(4-fluorophenoxy)propyl)-3-(4-trifluoromethylphenyl)guanidine(Compound F), 1-(3-(4-fluorophenoxy)propyl)-3-(4-chlorophenyl)guanidine(Compound G), a salt, solvate or N-oxide thereof, and any combinationsthereof.

The methods of the present invention may include certain embodiments. Inone embodiment, the Sigma receptor-related disease or disorder isselected from the group comprising cancer, neuropathic pain, depression,substance abuse, epilepsy, psychosis, Alzheimer's disease, Parkinson'sdisease, frontotemporal lobar degeneration, amyotrophic lateralsclerosis, and any combinations thereof. In another embodiment, thecancer is selected from the group consisting of prostate cancer, livercancer, pancreas cancer, CNS tumors, breast cancer, neuroblastoma,leukemia, and any combinations thereof. In yet another embodiment, thedisease or disorder is cancer and further wherein the administering ofthe therapeutic composition to the subject causes degradation of atleast one growth factor receptor in the subject's cancer. In yet anotherembodiment, the cancer comprises breast cancer or prostate cancer. Inyet another embodiment, the prostate cancer comprises castrate-sensitiveor castrate-insensitive prostate cancer. In yet another embodiment, theat least one growth factor receptor comprises EGFR, HER2, HER3, p95HER2,androgen receptor, and any combinations thereof. In yet anotherembodiment, the Sigma receptor is Sigma1. In yet another embodiment, thesubject is a mammal. In yet another embodiment, the mammal is a human.

The present invention also includes a method of preventing, treating orameliorating a Sigma receptor-related disorder or disease in a subjectin need thereof. The method comprises administering to the subject aneffective amount of a Sigma receptor-modulating compound, wherein themethod further comprises administering to the subject at least oneadditional therapeutic agent that inhibits the ubiquitin proteasomesystem (UPS) or autophagic survival pathway.

In one embodiment, the Sigma receptor-modulating compound is a Sigmareceptor antagonist. In another embodiment, the Sigma receptor isSigma1. In yet another embodiment, the Sigma receptor-modulatingcompound and the additional therapeutic agent are co-administered. Inyet another embodiment, the Sigma receptor-modulating compound and theadditional therapeutic agent are co-formulated. In yet anotherembodiment, the Sigma receptor-modulating compound and the additionaltherapeutic agent are administered at separate times. In yet anotherembodiment, administering the Sigma receptor-modulating compound to thesubject allows for administering a lower dose of the therapeutic agentto the subject, as compared to the dose of the therapeutic agent alonethat is required to achieve similar results in preventing, treating orameliorating the Sigma receptor-related disorder or disease in thesubject. In yet another embodiment, the Sigma-receptor related disorderor disease is cancer. In yet another embodiment, the cancer is selectedfrom the group consisting of prostate cancer, liver cancer, pancreascancer, breast cancer, neuroblastoma, CNS tumors, leukemia, and anycombinations thereof

In one embodiment, the Sigma receptor-modulating compound is selectedfrom the group consisting of:

(i) a compound of Formula (I):

wherein:

ring A is a monocyclic or bicyclic aryl or a monocyclic or bicyclicheteroaryl ring, and wherein the aryl or heteroaryl ring is optionallysubstituted with 0-4 R¹ groups;

each occurrence of R¹ is independently selected from the groupconsisting of —C₁-C₆ alkyl, —C₁-C₆ fluoroalkyl, —C₁-C₆ heteroalkyl, F,Cl, Br, I, —CN, —NO₂, —OR³, —SR³, —S(═O)R³, —S(═O)₂R³, —NHS(═O)₂R³,—C(═O)R³, —OC(═O)R³, —CO₂R³, —OCO₂R³, —CH(R³)₂, —N(R³)₂, —C(═O)N(R³)₂,—OC(═O)N(R³)₂, —NHC(═O)NH(R³), —NHC(═O)R³, —NHC(═O)OR³, —C(OH)(R³)₂, and—C(NH₂)(R³)₂;

each occurrence of R² is independently selected from the groupconsisting of H, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, and —C₁-C₃ alkyl-(C₃-C₆cycloalkyl), wherein the alkyl, heteroalkyl or cycloalkyl group isoptionally substituted with 0-5 R¹ groups, or X³ and R² combine to forma (C₃-C₇)heterocycloalkyl group, optionally substituted with 0-2 R¹groups;

each occurrence of R³ is independently selected from the groupconsisting of H, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, aryl, and —C₁-C₃alkyl-(C₃-C₆ cycloalkyl), wherein the alkyl, heteroalkyl, aryl, orcycloalkyl group is optionally substituted with 0-5 R¹ groups;

X¹ is —CH₂—, —S—, —O— or —(NR²)—;

X² is ═CH₂, ═S, ═O or ═NR²; and

X³ is —S—, —O—, or —NR²—; and

(ii) a compound of Formula (II):

R^(A)—R^(B)  (II), wherein;

R^(A) is selected from the group consisting of

X⁴ is selected from the group consisting of F, Cl, Br, and I; and

R^(B) is selected from the group consisting of:

(iii) haloperidol, IPAG, PB28, rimcazole, BD1063, BD1047, PRE84, NE100,(+)-SKF10047, HJH N(iv) a salt, solvate, or N-oxide thereof; andany combinations thereof.

In another embodiment, the compound of Formula (I) is selected from thegroup consisting of1-(3-(4-fluorophenoxy)propyl)-3-(4-iodophenyl)guanidine (Compound A),1-(3-(4-fluorophenoxy)propyl)-3-(4-methoxyphenyl)guanidine (Compound B),1-(n-propyl)-3-(4-iodophenyl)guanidine (Compound C),1-(n-propyl)-3-(4-methoxyphenyl)guanidine (Compound D),1-(3-(4-fluorophenoxy)propyl)-3-(4-trifluoromethylphenyl)guanidine(Compound F), 1-(3-(4-fluorophenoxy)propyl)-3-(4-chlorophenyl)guanidine(Compound G), a salt, solvate or N-oxide thereof, and any combinationsthereof.

In yet another embodiment, the compound of Formula (II) is selected fromthe group consisting of, 1,3-bis(3-(4-fluorophenoxy)propyl)guanidine(Compound E),1-(3-(4-fluorophenoxy)propyl)-3-(4-methyl-2-oxo-2H-chromen-7-yl)guanidine)(Compound H), a salt, solvate or N-oxide thereof, and any combinationsthereof.

In one embodiment, the Sigma receptor-modulating compound is selectedfrom the group consisting of:

(i) a compound of Formula (III):

wherein within Formula (III);

each occurrence of R¹ and R² is independently selected from the groupconsisting of —C₁-C₆ alkyl, —C₁-C₆ fluoroalkyl, —C₁-C₆ heteroalkyl, F,Cl, Br, I, —CN, —NO₂, —OR⁵, —SR⁵, —S(═O)R⁵, —S(═O)₂R⁵, —NHS(═O)₂R⁵,—C(═O)R⁵, —OC(═O)R⁵, —CO₂R⁵, —OCO₂R⁵, —CH(R⁵)₂, —N(R⁵)₂, —C(═O)N(R⁵)₂,—OC(═O)N(R⁵)₂, —NHC(═O)NH(R⁵), —NHC(═O)R⁵, —NHC(═O)OR⁵, —C(OH)(R⁵)₂, and—C(NH₂)(R⁵)₂;

R³ is selected from the group consisting of —C₁-C₆ alkyl, —C₁-C₆fluoroalkyl, —C₁-C₆ alkoxy, F, Cl, Br, and I;

R⁴ is selected from the group consisting of —C₁-C₆ alkyl, —C₁-C₆ alkoxy,F, Cl, Br, and I;

each occurrence of R⁵ is independently selected from the groupconsisting of H, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, aryl, and —C₁-C₃alkyl-(C₃-C₆ cycloalkyl), wherein the alkyl, heteroalkyl, aryl, orcycloalkyl group is optionally substituted;

X is selected from the group consisting of CH₂, C═O, or O;

n is an integer from 1-3;

x is an integer from 0-4; and

y is an integer from 0-4;

(ii) haloperidol, IPAG, PB28, rimcazole, BD1063, BD1047, PRE084, NE100,(+)-SKF10047, (+)-pentazocine;(iii) a salt, solvate, or N-oxide thereof; andany combinations thereof.

In one embodiment, the compound of Formula (III) is selected from thegroup consisting of1-(3-(4-fluorophenoxy)propyl)-3-(4-iodophenyl)guanidine (Compound A),1-(3-(4-fluorophenoxy)propyl)-3-(4-methoxyphenyl)guanidine (Compound B),1-(3-(4-fluorophenoxy)propyl)-3-(4-trifluoromethylphenyl)guanidine(Compound F), 1-(3-(4-fluorophenoxy)propyl)-3-(4-chlorophenyl)guanidine(Compound G), a salt, solvate or N-oxide thereof, and any combinationsthereof.

In one embodiment, the therapeutic agent is selected from the groupconsisting of growth factor receptor inhibitors, monoclonal antibodiesagainst growth factor receptors, hormone receptor antagonists, autophagymodulators, ER stress response inhibitors, proteasome inhibitors, andany combinations thereof.

In one embodiment, the therapeutic agent is selected from the groupconsisting of octapeptide, somatostatin, analoguem, lanreotide,angiopeptin, dermopeptin, octreotide, pegvisomant, 3-methyladenine,chloroquine, hydroxychloroquine, wortmannin, eeyarestatin I, salubrinal,versipelostatin, 2H-isoindole-2-carboxylic acid, 4-fluoro-1,3-dihydro-,(2R,6S, 12Z, 13aS,14aR,16aS)-14a-[[(cyclopropylsulfonyl)amino]carbonyl]-6-[[(1,1-dimethylethoxy)carbonyl]amino]-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a-hexadecahydro-5,16-dioxocyclopropa[e]pyrrolo[1,2-a][1,4]diazacyclopentadecin-2-ylester (Danoprevir),adamantane-acetyl-(6-aminohexanoyl)₃-(leucinyl)₃-vinyl-(methyl)-sulfone,N-acetyl-L-leucyl-L-leucyl-L-methional,N-[(phenylmethoxy)carbonyl]-L-leucyl-N-[(1S)-1-formyl-3-methylbutyl]-L-leucinamide,(2R,3S,4R)-3-hydroxy-2-[(1S)-1-hydroxy-2-methylpropyl]-4-methyl-5-oxo-2-pyrrolidinecarboxy-N-acetyl-L-cysteinethioester, N—[N—(N-acetyl-L-leucyl)-L-leucyl]-L-norleucine, lactacystin,4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride,(S)-1-carboxy-2-phenyl]-carbamoyl-arg-val-arginal, bovine pancreatictrypsin inhibitor, [(2S,2R)-3-amino-2-hydroxy-4-phenylbutanoyl]-L-leucine,N—[(S)-1-carboxy-isopentyl)-carbamoyl-alpha-(2-iminohexahydro-4-(S)-pyrimidyl]-L-glycyl-L-phenylalaninal,ethylenediamine-tetraacetic acid disodium salt dehydrate,acetyl-leucyl-leucyl-arginal, isovaleryl-val-val-AHMHA-ala-AHMHA whereAHMHA=(3S, 4S)-4-amino-3-hydroxy-6-methylheptanoic acid,N-alpha-L-rhamnopyranosyloxy (hydroxyphosphinyl)-L-leucyl-L-tryptophan,phenylmethanesulfonyl fluoride, bortezomib, carfilzomib, ONX 0912,NPI-0052, CEP-18770, MLN9708, disulfiram, epigallocatechin-3-gallate,salinosporamide A, PI3K inhibitors, lapatinib, rapamycin, rapalogs, heatshock protein (HSP) inhibitors, androgen receptor inhibitors,conjugation products of Sigma ligands with targeting components, a saltthereof, and any combinations thereof.

In one embodiment, the subject is a mammal. In another embodiment, themammal is a human.

The present invention also includes a method of modulating cellularprotein homeostasis in a subject in need thereof. The method comprisesadministering to the subject an effective amount of a Sigmareceptor-modulating compound, whereby cellular protein homeostasis inthe subject is modulated.

In one embodiment, the Sigma receptor-modulating compound is a Sigmareceptor antagonist. In another embodiment, the Sigma receptor isSigma1. In yet another embodiment, the Sigma receptor-modulatingcompound and the additional therapeutic agent are co-administered. Inyet another embodiment, the Sigma receptor-modulating compound and theadditional therapeutic agent are co-formulated. In yet anotherembodiment, the Sigma receptor-modulating compound and the additionaltherapeutic agent are administered at separate times.

In one embodiment, the Sigma receptor-modulating compound is selectedfrom the group consisting of:

(i) a compound of Formula (I):

wherein:

ring A is a monocyclic or bicyclic aryl or a monocyclic or bicyclicheteroaryl ring, and wherein the aryl or heteroaryl ring is optionallysubstituted with 0-4 R¹ groups;

each occurrence of R¹ is independently selected from the groupconsisting of —C₁-C₆ alkyl, —C₁-C₆ fluoroalkyl, —C₁-C₆ heteroalkyl, F,Cl, Br, I, —CN, —NO₂, —OR³, —SR³, —S(═O)R³, —S(═O)₂R³, —NHS(═O)₂R³,—C(═O)R³, —OC(═O)R³, —CO₂R³, —OCO₂R³, —CH(R³)₂, —N(R³)₂, —C(═O)N(R³)₂,—OC(═O)N(R³)₂, —NHC(═O)NH(R³), —NHC(═O)R³, —NHC(═O)OR³, —C(OH)(R³)₂, and—C(NH₂)(R³)₂;

each occurrence of R² is independently selected from the groupconsisting of H, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, and —C₁-C₃ alkyl-(C₃-C₆cycloalkyl), wherein the alkyl, heteroalkyl or cycloalkyl group isoptionally substituted with 0-5 R¹ groups, or X³ and R² combine to forma (C₃-C₇)heterocycloalkyl group, optionally substituted with 0-2 R¹groups;

each occurrence of R³ is independently selected from the groupconsisting of H, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, aryl, and —C₁-C₃alkyl-(C₃-C₆ cycloalkyl), wherein the alkyl, heteroalkyl, aryl, orcycloalkyl group is optionally substituted with 0-5 R¹ groups;

X¹ is —CH₂—, —S—, —O— or —(NR²)—;

X² is ═CH₂, ═S, ═O or ═NR²; and

X³ is —S—, —O—, or —NR²—; and

(ii) a compound of Formula (II):

R^(A)—R^(B)  (II), wherein;

R^(A) is selected from the group consisting of

X⁴ is selected from the group consisting of F, Cl, Br, and I; and

R^(B) is selected from the group consisting of:

(iii) haloperidol, IPAG, PB28, rimcazole, BD1063, BD1047, PRE084, NE100,(+)-SKF10047, (+)-pentazocine;(iv) a salt, solvate, or N-oxide thereof; andany combinations thereof.

In another embodiment, the compound of Formula (I) is selected from thegroup consisting of1-(3-(4-fluorophenoxy)propyl)-3-(4-iodophenyl)guanidine (Compound A),1-(3-(4-fluorophenoxy)propyl)-3-(4-methoxyphenyl)guanidine (Compound B),1-(n-propyl)-3-(4-iodophenyl)guanidine (Compound C),1-(n-propyl)-3-(4-methoxyphenyl)guanidine (Compound D),1-(3-(4-fluorophenoxy)propyl)-3-(4-trifluoromethylphenyl)guanidine(Compound F), 1-(3-(4-fluorophenoxy)propyl)-3-(4-chlorophenyl)guanidine(Compound G), a salt, solvate or N-oxide thereof, and any combinationsthereof.

In yet another embodiment, the compound of Formula (II) is selected fromthe group consisting of, 1,3-bis(3-(4-fluorophenoxy)propyl)guanidine(Compound E),1-(3-(4-fluorophenoxy)propyl)-3-(4-methyl-2-oxo-2H-chromen-7-yl)guanidine)(Compound H), a salt, solvate or N-oxide thereof, and any combinationsthereof.

In one embodiment, the Sigma receptor-modulating compound is selectedfrom the group consisting of:

(i) a compound of Formula (III):

wherein within Formula (III);

each occurrence of R¹ and R² is independently selected from the groupconsisting of —C₁-C₆ alkyl, —C₁-C₆ fluoroalkyl, —C₁-C₆ heteroalkyl, F,Cl, Br, I, —CN, —NO₂, —OR⁵, —SR⁵, —S(═O)R⁵, —S(═O)₂R⁵, —NIHS(═O)₂R⁵,—C(═O)R⁵, —OC(═O)R⁵, —CO₂R⁵, —OCO₂R⁵, —CH(R⁵)₂, —N(R⁵)₂, —C(═O)N(R⁵)₂,—OC(═O)N(R⁵)₂, —NHC(═O)NH(R⁵), —NHC(═O)R⁵, —NHC(═O)OR⁵, —C(OH)(R⁵)₂, and—C(NH₂)(R⁵)₂;

R³ is selected from the group consisting of —C₁-C₆ alkyl, —C₁-C₆fluoroalkyl, —C₁-C₆ alkoxy, F, Cl, Br, and I;

R⁴ is selected from the group consisting of —C₁-C₆ alkyl, —C₁-C₆ alkoxy,F, Cl, Br, and I;

each occurrence of R⁵ is independently selected from the groupconsisting of H, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, aryl, and —C₁-C₃alkyl-(C₃-C₆ cycloalkyl), wherein the alkyl, heteroalkyl, aryl, orcycloalkyl group is optionally substituted;

X is selected from the group consisting of CH₂, C═O, or O;

n is an integer from 1-3;

x is an integer from 0-4; and

y is an integer from 0-4;

(ii) haloperidol, IPAG, PB28, rimcazole, BD1063, BD1047, PRE084, NE100,(+)-SKF10047, (+)-pentazocine;(iii) a salt, solvate, or N-oxide thereof; andany combinations thereof.

In one embodiment, the compound of Formula (III) is selected from thegroup consisting of1-(3-(4-fluorophenoxy)propyl)-3-(4-iodophenyl)guanidine (Compound A),1-(3-(4-fluorophenoxy)propyl)-3-(4-methoxyphenyl)guanidine (Compound B),1-(3-(4-fluorophenoxy)propyl)-3-(4-trifluoromethylphenyl)guanidine(Compound F), 1-(3-(4-fluorophenoxy)propyl)-3-(4-chlorophenyl)guanidine(Compound G), a salt, solvate or N-oxide thereof, and any combinationsthereof.

In one embodiment, the subject is afflicted with a neurodegenerativedisease. In another embodiment, the neurodegenerative disease comprisesParkinson's disease, frontotemporal lobar degeneration, amyotrophiclateral sclerosis, or any combinations thereof. In yet anotherembodiment, the subject is a mammal. In yet another embodiment, themammal is a human.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of theinvention are better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings embodiments that are presently preferred. Itshould be understood, however, that the invention is not limited to theprecise arrangements and instrumentalities of the embodiments shown inthe drawings.

FIGS. 1A-1B illustrate prostate adenocarcinoma cell death induced bySigma1 antagonists. FIG. 1A is a graph illustrating Sigmaligand-mediated decrease in cell number measured by Alamar blue assay:dose-response analysis of DU145 cells treated with Sigma antagonist(haloperidol) or agonist (PRE084) for 3 days and compared to control(DMSO treated). FIG. 1B is a graph illustrating PC3 cell deathquantitated by trypan blue exclusion assay following treatment with 5 μMSigma antagonist (haloperidol) or agonist (PRE084) for up to three daysand compared to control (DMSO treated) cell culture. Quantitated celldeath was presented as the percentage of trypan blue positive cells perdrug treated population. Salient cell death occurred following ˜24-48hours of treatment with the Sigma antagonist.

FIGS. 2A-2B are a set of flowcharts illustrating a model for Sigmaantagonist-mediated tumor cell death. FIG. 2A is a flowchartillustrating that Sigma antagonists induce endoplasmic reticulum (ER)stress, resulting in a sequence of adaptive or cytoprotective responsescomprising UPR and autophagy, wherein UPR induces autophagy as acytoprotective response. FIG. 2B is a flowchart illustrating that, whenthe cytoprotective response of autophagy is overwhelmed, the cellundergoes apoptosis.

FIG. 3 is a scheme illustrating a size and volume comparison of the arylguanidine of IPAG to the 4-phenylpiperidine moiety of haloperidol.

FIGS. 4A-4I illustrate autophagosome formation and autophagic fluxassociated with Sigma antagonist treatment. FIG. 4A is an image of a gelillustrating the finding that treatment of MDA-MB-468 breastadenocarcinoma cells for 24 hours with 10M Sigma antagonists (IPAG,haloperidol, PB28, rimcazole), but not agonists (PRE-084, (+)-SKF10047,(+)-pentazocine), resulted in a salient induction of LC3II levels. Sizemarkers indicate kilodaltons (kDa). FIG. 4B is a series of photographsillustrating translocation of GFP-tagged LC3 (GFP-LC3) intoautophagosomes in Sigma ligand treated MDA-MB-468(GFP-LC3) cells. Cellswere treated for 24 hours with 10 μM IPAG, haloperidol, PB28, rimcazole,and with 50 μM PRE-084, (+)-SKF10047, (+)-pentazocine. (+)-SKF10047 isabbreviated as (+)-SKF and (+)-pentazocine is abbreviated as (+)-PTZ.FIG. 4C is a graph illustrating dose-responsive translocation of GFP-LC3into autophagosomes. GFP-LC3 punctae were quantitated inMDA-MB-468(GFP-LC3) cells treated for 24 hours with increasingconcentrations of antagonists and agonists. Histograms represent datafrom at least four determinations, presented as the mean number+S.E.M ofpunctae per cell at the indicated doses of drug. Drug EC₅₀+S.E.M. valuesare indicated. Data are representative of at least 10 fields and 300cells for each drug concentration. P<0.001 for all antagonists comparedto agonists; P<0.05 for IPAG and haloperidol compared to PB28 andrimcazole. FIG. 4D is an image of a gel illustrating accumulation ofLC3II in presence of E64d. Cell lysates were evaluated after 18 hours ofcotreatment of E64d (20 μg/ml) with IPAG (10 μM). FIG. 4E is a graphillustrating effects of haloperidol (10 μM) combined with E64d. FIG. 4Fis a graph illustrating effects of IPAG combined with E64d. Thehistograms in FIGS. 4E-4F illustrate quantitations of the ratio of LC3IIband density in treated over DMSO control lanes, and the data arepresented as the fold induction in LC3II band density compared tocontrol (DMSO alone) (n=4, *P<0.05). Note that no histogram bar is shownfor DMSO alone as this condition is set as the control, baseline. FIG.4G a gel illustrating that cleavage of GFP-LC3 was used to furtherevaluate autophagic flux. MDA-MB-468(GFP-LC3) cells were treated for 24hours with the indicated Sigma ligands. FIG. 4H is an image of a gelillustrating that there was no accumulation of GFP-LC3II in the absenceof IPAG and E64d. FIG. 4I is an image of a gel illustrating accumulationof GFPLC3II and diminished GFP by combined treatment with IPAG (10 μM)and E64d (20 μg/ml).

FIGS. 5A-5C illustrate the finding that Sigma1 antagonist-mediatedautophagy is Sigma1 dependent. MDA-MB-468(GFP-LC3) cells were treatedfor 24 hours with 10 μM IPAG, 72 hours following transfection witheither control or Sigma1 siRNA. FIG. 5A is an image of an immunoblotconfirming that siRNA-mediated knockdown of Sigma1 was evaluatedpost-transfection and post-treatment in MDA-MB-468(GFP-LC3) cells.Levels of GFP-LC3 cleavage were determined by GFP immunoblot. Data arerepresentative of three determinations. FIG. 5B is a representativeimage of GFP-LC3 puncta formation in MDA-MB-468(GFP-LC3) cells treatedfor 24 with 10 μM IPAG. FIG. 5C is a graph illustrating quantitation ofGFP-LC3 puncta formation in MDA-MB-468(GFP-LC3) cells treated for 24with 10 μM IPAG.

FIGS. 6A-6F illustrate dose-responsive induction of UPR by Sigma1antagonist. MDA-MB-468 cells were treated for 24 hours with increasingdoses of IPAG (1 to 20 μM). FIG. 6A is an image of a gel illustratinginduction of LC3II protein levels. FIG. 6B is an image of a gelillustrating induction of GRP78/BiP protein levels. FIG. 6C is an imageof a gel illustrating phosphorylation of p38MAPK (Thr180/Tyr182). FIG.6D is an image of a gel illustrating induction of IRE1α protein levelsand phosphorylation of JNK (Thr183/Tyr185). FIG. 6E is an image of a gelillustrating induction of ATF4 protein levels and phosphorylation ofeIF2α (Ser51). FIG. 6F is a graph illustrating quantification ofautophagosomes and UPR marker induction following 24-hour treatment with1 μM IPAG. Data generated from at least three independentdeterminations, and are presented as mean fold induction over DMSOtreated control. Error bars represent the standard error of the mean.*P<0.05, ***P<0.001.

FIGS. 7A-7E illustrate the time-course of Sigma1 antagonist-induced UPR.Time-course of Sigma1 antagonist-induced ER stress was evaluated byimmunoblot analysis of UPR markers. Cells were treated for indicatedtimes with 10 μM IPAG. FIG. 7A is an image of a gel illustratinginduction of IRE1α protein levels and phosphorylation of JNK(Thr183/Tyr185). FIG. 7B is an image of a gel illustrating induction ofATF4 protein levels and phosphorylation of eIF2α (Ser51). FIG. 7C is animage of a gel illustrating phosphorylation of p38MAPK (Thr180/Tyr182).FIG. 7D is an image of a gel illustrating induction of GRP78/BiP proteinlevels. FIG. 7E is an image of a graph illustrating time-actionhistogram of autophagosome formation in MDA-MB-468(GFP-LC3). Data arerepresentative of at least 10 fields and 300 cells for each drugconcentration. P<0.001 for 24 hour IPAG treatment compared to 0 hour(basal), 1 hour, and 6 hour; P<0.05 for 24 hour compared to 12 hour IPAGtreatment.

FIGS. 8A-8D illustrate the finding that the inhibition of Sigma1antagonist mediated UPR inhibits autophagy. FIG. 8A is an image of animmunoblot illustrating IRE1α siRNA knockdown, 48-72 hourspost-transfection. Knockdown of IRE1a abrogates IPAG mediated inductionof GFP-LC3 cleavage. FIG. 8B comprises representative images of siRNAmediated knockdown of IRE1α and ATF4 abrogate GFP-LC3 punctae formation.FIG. 8C is a graph quantifying siRNA mediated knockdown of IRE1α and theabrogation of GFP-LC3 punctae formation by ATF4. FIG. 8D is a graphillustrating that JNK inhibitor, SP610250, abrogates IPAG mediatedpunctae formation. MDA-MB-468(GFP-LC3) cells were treated for 24 hourswith a combination of IPAG (10 μM) and JNK inhibitor SP610250 (20 μM).

FIGS. 9A-9C illustrate inhibition of Sigma1 antagonist-associatedautophagy by Beclin1 RNAi. Beclin1 or control siRNA was transfected 72hours prior to treatment with Sigma1 antagonists. Cells were treated for24 hours with 10 μM of the indicated Sigma1 antagonists (IPAG,haloperidol). FIG. 9A illustrates representative images ofMDA-MB-468(GFP-LC3) cells treated as described above. FIG. 9B is a graphillustrating quantification of images as mean number of GFP-LC3 punctaeper cell. Data are representative of at least 10 fields and 200 cellsfor each treatment condition. FIG. 9C is an image of an immunoblotconfirming siRNA knockdown of Beclin1 and inhibition of Sigma1antagonist-mediated GFP-LC3 cleavage in Beclin1 knockdown cells.

FIGS. 10A-10F illustrate the finding that inhibition of UPR or autophagyaccelerates Sigma antagonist-induced apoptosis. FIG. 10A is a graphillustrating the time-course of Sigma1 antagonist-induced cell death.MDA-MB-468 cells were treated for 24 and 48 hours with 10 μM IPAG(antagonist), and compared to cells treated with 10 μM PRE-084 (agonist)or DMSO (control) for the same time period. Cell death is measured bytrypan blue exclusion, and is presented as the percentage of dead cellsin a counted population. Data are from at least 4 determinations(***P<0.001). FIG. 10B is an image of an immunoblot demonstratingCaspase 3 (Asp 175) cleavage (cCaspase) following 48 hours of IPAGtreatment. PRE-084 is abbreviated as PRE. FIG. 10C is a graphillustrating Sigma1 antagonist (IPAG) induced cell death in Beclin1knockdown cells, 72 hours following transfection of Beclin1 siRNA. Dataare from at least 4 determinations. FIG. 10D is an image of animmunoblot confirming siRNA mediated Beclin1 knockdown and demonstratingIPAG induced apoptosis by cleavage of Caspase 3 (cCaspase) and PARP(cPARP). FIG. 10E illustrates Sigma1 antagonist-induced cell deathmeasured in IRE1α knockdown cells. Cells were treated for 24 hours with10 μM IPAG, 72 hours following transfection of IRE1α siRNA. Cell deathwas quantitated as in FIG. 10A. FIG. 10F is an image of an immunoblotillustrating apoptotic cell death was confirmed by immunoblot detectionof cleaved Caspase 3 (cCaspase) and cleaved PARP (cPARP) as in FIG. 10D.

FIG. 11 is a graph illustrating decrease in MDAMB468 cell sizeassociated with the Sigma1 antagonist treatment. MDA-MB-468 cells weretreated for 24 hours with DMSO (control), 10 μM IPAG (antagonist), or 10μM PRE084 (agonist). Cell size was quantitated by flow cytometry, usinga Becton Dickinson FACS Calibur flow cytometer with Cell Quest software.The mean forward scatter height (FSC-H) of the G1-phase population wasdetermined as a measure of relative cell size. Single cells were gatedaway from aggregated cells using an FL2-width versus FL-2 area dot plot.Mean FSC-H+S.E. was calculated from at least 4 independentdeterminations. For each FSC-H determination, FACS analysis wasperformed on 10,000 single cells. The mean FSC-H of DMSO (control) andPRE084 treated MDA-MB-468 measured 370±6 and 372±7, respectively,whereas the mean FSC-H of IPAG treated MDA-MB-468 cells was 323±4. **P<0.01 for IPAG compared to DMSO and IPAG compared to PRE084.

FIGS. 12A-12B illustrate the finding that chemical inhibition ofautophagy accelerates Sigma1 antagonist mediated cell death. MDA-MB-468cells were treated for 24 hours with DMSO (control), 10 μM IPAG (Sigmaantagonist), or 5 mM 3-methyladenine (3-MA, autophagy inhibitor), or acombination of IPAG and 3-MA. FIG. 12A is a graph illustratingquantitation of GFP-LC3 punctae per cell. Data were quantitated fromfour determinations, and are presented as mean+S.E.M. FIG. 12B is agraph illustrating cell death measured by trypan blue exclusion. Celldeath, above control levels, was observed at 24 hours only when the IPAGand 3-MA were combined. Statistical significance was determined byone-way ANOVA, followed by Bonferroni's Post-test.

FIGS. 13A-13B describe Sigma1 receptor-associated proteins. Experimentsinvolved protein complex purification and identification experimentswith dual affinity-tagged Sigma1 with tandem hemagglutin (HA) epitopeand six-histidine (His₆). FIG. 13A is an image of a silver stain gelillustrating Sigma1-HA-His₆ associated proteins isolated bytandem-affinity purification from MDA-MB-468 breast adenocarcinomacells. Approximately 80 proteins associated (i.e., co-purified) withSigma1. FIG. 13B is a pie chart illustrating Sigma1-associated proteinprofile determined by MUD-Pit LC-MS/MS. 80% of Sigma1-associatedproteins were involved in cellular processes directly relevant to ERprotein homeostatis, cell survival, and death.

FIG. 14 is an image of an immunoblot illustrating the increased level ofubiquinated proteins associated with Sigma antagonist treatment. PC3prostate adenocarcinoma cells treated with 10 μM haloperidol(antagonist) or DMSO (control). Total cell lysates were resolved bySDS-PAGE and immunoblotted with ubiquitin antibody (P4D1). Similarresults were obtained with DU145 prostate cancer cells.

FIG. 15 is an image of an immunoblot illustrating induction of UPR in aprostate cancer cell line. The immunoblot is of cell detergent solublecell lysate from PC3 prostate adenocarcinoma cells treated for 20 hourswith 10 μM haloperidol (antagonist) and compared to DMSO (control)treated cells. P-IRE1α indicates phosphorylated IRE1α. Similar resultswere obtained with DU145 prostate cancer cells.

FIGS. 16A-16B illustrate Sigma antagonist associated autophagy. FIG. 16Aillustrates representative images of the translocation of GFP-tagged LC3(LC3-GFP) into autophagosomes in a control (DMSO) group and ahaloperidol-treated group (10 μM, antagonist).

FIG. 16B is an image of a gel illustrating increased levels of themicrotubule-associated protein light chain 3BII isoform (CL3BII), whichis a broadly used indicator of activated autophagy. Treatment with 10 μMof haloperidol for 24 hours resulted in a salient induction of LC3BIIlevels.

FIG. 17 is a synthetic scheme illustrating a generalized synthetic routetoward novel hybrid IPAG-Haloperidol analogs

FIG. 18 is a synthetic scheme illustrating an alternative synthesis ofN-aryl-N′-substituted guanidines.

FIG. 19 is a synthetic scheme illustrating a synthesis of radio-labeledhybrid IPAG-haloperidol.

FIG. 20 is a synthetic scheme illustrating a generalized synthetic routetoward N-aryl-N′-substituted guanidines.

FIG. 21 is a synthetic scheme illustrating an alternative generalizedsynthetic route toward N-aryl-N′-substituted guanidines.

FIG. 22 is a synthetic scheme illustrating a general synthetic route toguanidine precursors using solution or solid phase methods.

FIG. 23 is a synthetic scheme illustrating the synthesis of haloperidolamine.

FIG. 24 is a synthetic scheme illustrating the synthesis ofhaloperidol-like amines.

FIG. 25 is a list of representative structures of guanidine Sigmaantagonists, with variable groups substituted at R¹.

FIGS. 26A-26B illustrate six prototypical Sigma receptor ligands. FIG.26A is a list illustrating the six prototypic Sigma receptor ligandsselected for their selectivity, Sigma1 or Sigma2 or both, and theirputative pharmacological activity as an agonist or antagonist. FIG. 26Billustrates the corresponding chemical structures of the Sigma receptorligands. All compounds shown here are commercially available.

FIGS. 27A-27C illustrates the finding that Sigma receptor antagonistsinhibit proliferation of both estrogen receptor-positive and -negativecells. In vitro cell proliferation was quantitated by Alamar bluereduction. The anti-proliferative effects of six drug concentrations ofthe indicated drugs were quantitated after 4 days of treatment. In thesebreast adenocarcinoma cell cultures, Sigma1 agonists (+)-pentazocine and(+)-SKF10047 (data shown for the latter) were ineffective atconcentrations as high as 0.1M. Sigma1 antagonists IPAG and rimcazoleinhibited cell proliferation with comparable potencies in all celllines. FIG. 27A is a graph illustrating that tamoxifen, IPAG, andrimcazole inhibited cell proliferation with similar potencies in theestrogen receptor positive breast tumor cell (MCF-7) culture. FIG. 27Bis a graph illustrating that tamoxifen, IPAG, and rimcazole inhibitedcell proliferation with similar potencies in the estrogen receptorpositive breast tumor cell (T47D) culture. FIG. 27C is a graphillustrating that the Sigma antagonists IPAG and rimcazole alsoinhibited cell proliferation of the estrogen receptor negativeMDA-MB-468 cells with similar potencies. Other Sigma1 antagonists,BD1063, BD1047, and haloperidol also inhibited cell proliferation (datanot shown). Data are representative of 3 experiments performed induplicate.

FIG. 28 is a graph illustrating the finding that a Sigma receptorantagonist potentiates tumor growth inhibition by anti-estrogen therapy.Tamoxifen potentiation by rimcazole was evaluated in vivo. Preliminarytumor xenograft experiments were performed according to MSKCC TumorAssessment Core facility protocol. Briefly, β-estradiol treated athymicmice were injected with MCF7 cells, and drug treatment was initiatedwhen mean tumor volume reached approximately 140 mm³, which occurred 17days following MCF-7 injection. Daily i.p. drug injections wereperformed for up to 11 days, and tumor volume was quantified by theformula: tumor volume=L×W²×π/6. In this pilot experiment, 3-5 mice weretested per group. After 11 days of drug treatment, tamoxifen (1 mg/kg)and Rimcazole (10 mg/kg) had each inhibited tumor growth byapproximately 50%. During the 11 day time course, tumor growth wascompletely inhibited when tamoxifen (1 mg/kg) and Rimcazole (10 mg/kg)were combined. An extended time course, with more mice per group andmultiple drug doses, may be performed to determine whether the tumorgrowth inhibitory effect of combined drug treatment is additive orsynergistic.

FIG. 29 illustrates the time-course of a Sigma1 antagonist inducingapoptotic cell death of breast adenocarcinoma cells. Treatment withSigma1 antagonist, IPAG, induces apoptotic cell death of MDA-MB-468cells, as indicated by the percentage of subG1 phase cells. MDA-MB-468cell treated for 24 and 48 hours with 10 μM IPAG (Sigma1 ligand,putative antagonist) showed a significant increase in the number ofsubG1 (dead, apoptotic) cells after 48 h of treatment, 31.7% (indicatedby M1 cell population). The far left panel represents cells treated or24 hours with DMSO (vehicle control).

FIGS. 30A-30B illustrate that Sigma1 antagonists mediate cell cyclearrest. Treatment with Sigma1 antagonist, IPAG, results in accumulationof breast adenocarcinoma cells in G1 phase of the cell cycle. FIG. 30Ais a graph illustrating the percentage of G1, S, and G2 phase cells inT47D cells treated for 24 hours with DMSO (vehicle control), 1 μM IPAG(Sigma1 antagonist), or 10 μM (+)SKF-10047 (Sigma1 agonist). FIG. 30 Bis a graph illustrating the percentage of G1, S, and G2 phase cells inMDA-MB-468 cells treated for 24 hours with DMSO (vehicle control), 10 μMIPAG (Sigma1 antagonist), or 10 μM (+)-SKF-10047 (Sigma1 agonist). Incontrast to IPAG treatment, control (DMSO) and (+)-SKF10047 (SKF)treated cells did not present significant difference in cell cycleprofile.

FIGS. 31A-31E show that Sigma1 antagonists diminish tumor cell size in adose and time responsive manner. FIG. 31A is a differential interferencecontrast (DIC) image of MDA-MB-468 breast adenocarcinoma cells treatedfor ˜20 hours with DMSO (drug vehicle control), 10 μM IPAG (antagonist)or 10 μM PRE084 (agonist). A twenty-micron bar is shown in each image.Data are presented as mean±SEM. FIG. 31B is a graph illustrating thefinding that IPAG diminished the mean cell size (FSC-H) of MDA-MB-468breast tumor cells in a time-responsive manner. The mean FSC-H of 370±5at to decreased to 323±4 after 24 hours of IPAG treatment and to 267±2after 48 hours of treatment. Two-tailed t-tests were performed todetermine statistical significance. For IPAG treatment compared tocontrol (DMSO) and Sigma agonists (SKF and PRE084), P=0.0002 at d1 andP=0.00013 at d2. Data were generated from three to five independentdeterminations for each time-point. Flow cytometry was used to measuremean cell size (mean FSC-H) of T-47D breast adenocarcinoma cells. FIG.31C is a bar graph illustrating T-47D cells treated for 24 hours with 10μM indicated Sigma ligand. IPAG and BD 1047 (Sigma1 antagonists)diminished cell size from a mean FSC-H of 412±5 for control (DMSO) cellsversus 341±7 and 381±8 for IPAG and BD1047 treated cells, respectively.Both Sigma1 putative agonists, (+)-SKF10047 (SKF) and PRE-084, did notalter cell size. Data represent 5 independent determinations (*P<0.05,***P<0.001). FIG. 31D is a bar graph illustrating how IPAG (antagonist)decreases cell size in a dose responsive manner. T-47D were treated for24 h with 1 and 10 μM IPAG, and mean FSC-H was measured. FIG. 31E is agraph illustrating that T47D cell size decreases over time. T-47D cellswere treated for a total of 48 hours with 10 μM of the indicated Sigmaligands. IPAG treatment decreased the mean FSC-H of G1 phase T-47D from412±5 to 331±3 and 300±2 at 24 hours (d1) and 48 hours (d2) of drugtreatment. Neither Sigma1 agonist, (+)-SKF10047 and PRE084, altered cellsize. These data were generated from at least three independentexperiments. Two-tailed t-tests were performed to determine statisticalsignificance. For IPAG treatment compared to control (DMSO) and Sigma1agonists (SKF and PRE084), P=0.0002 at d1 and P<0.0001 at d2. For FIGS.31B-31E, the mean FSC-H was determined for each cell cycle population(i.e., G1, S, G2/M), and data is shown for G1 phase cells. S and G2/Mphase cells responded similarly (data not shown).

FIGS. 32A-32B illustrate induction of unfolded protein response (UPR)associated with Sigma1 antagonist treatment. FIG. 32A comprises imagesof immunoblots of MDA-MB-468 breast adenocarcinoma cells treated with 10μM Sigma1 antagonist (IPAG) for the indicated times, up to 24 hours.Total cell lysates were resolved by SDS-PAGE and immunoblotted for UPRmarkers IRE1α, GRP78/BiP, GRP94, and ORP150. The higher migrating IRE1αband was consistent with the phosphorylated form (P-IRE1α). FIG. 32B isa series of images of immunoblots illustrating a range of tumor celllines treated for ˜24 hours with 10 μM IPAG (Sigma1 antagonist) or 10 μMPRE084 (Sigma1 agonist). Increased levels of GRP78/BiP (BiP) was anindicator of UPR. Cell lines include: breast adenocarcinoma (MDAMB468,MCF-7, T47D), prostate adenocarcinoma (DU145, PC3), hepatocellularcarcinoma (HepG2), pancreatic adenocarcinoma (Panc1).

FIGS. 33A-33D illustrate the finding that Sigma1 antagonist treatmentmediated translation arrest. FIG. 33A is an image of an immunoblot ofT-47D cells treated for 20 hours with 10 μM of Sigma1 antagonists, IPAGand haloperidol (HPL), or the agonists, PRE-084 and (+)SKF10047,followed by a 1 hour pulse with [³⁵S]-methionine and cysteine to measurenew protein synthesis. Detergent soluble protein extracts were resolvedby 10% SDS-PAGE, transferred onto PVDF filter and analyzed for[³⁵S]-labeled protein content following 3 day exposure to autoradiographfilm and subsequently immunoblotted to detect Sigma1 and 3 actin levels.FIG. 31B is an image of an immunoblot of T-47D cells treated for 20hours with 10 μM of the indicated Sigma ligands, IPAG (antagonist) andPRE084 (agonist); levels of phosphothreonine 389-p70S6Kinase (P-S6K) andphosphoserine 65-4E-BP1 (P-4E-BP1) were evaluated by immunoblot. FIG.33C is an image of an immunoblot of the time-course of changes in P-S6Kand P-4E-BP1 levels in response to treatment with 10 μM IPAG for theindicated time periods. FIG. 33D is an image of an immunoblotillustrating that translation arrest was further confirmed by immunoblotdetection of phospho-serine 209-eIF4E (P-eIF4E) and phosphoserine51-eIF2α (P-eIF2α) under the same conditions described in FIG. 33Cabove.

FIGS. 34A-34B illustrate the finding that Sigma1 antagonist treatment isassociated with an increased level of ubiquitylated proteins. FIG. 34Ais an image of an immunoblot of MDA-MB-468 breast adenocarcinoma cellstreated with 10 μM Sigma1 antagonist (IPAG) for the indicated times, upto 24 hours. Total cell lysates were resolved by SDS-PAGE andimmunoblotted with a poly-ubiquitin antibody P4D1. FIG. 34B is an imageof an immunoblot of HepG2 hepatocellular carcinoma cells treated for 20hours with 10 μM of indicated Sigma1 ligands. Immunoblots revealedincreased levels of ubiquitylated proteins (detected with the P4D1anti-poly-ubiquitin antibody).

FIGS. 35A-35C illustrate the finding that treatment with haloperidolresulted in elevated levels of poly-ubiquitylated proteins andtranslation arrest in subcortical structures. Subcortical region(subcortical) of Balb-c mouse brain dissected following 24 hourtreatment with 10 mg/kg haloperidol (Haldol), injectedintraperitoneally. FIG. 35A is an image of an immunoblot illustratingincreased levels of poly-ubiquitylated (poly-Ub) proteins in response tohaloperidol (Haldol) treatment. FIG. 35B is an image of an immunoblotillustrating translation arrest markers phospho-Ser 209-elF4E (P-eIF4E)and phosphoThr 389-p70S6Kinase (P-S6K). FIG. 35C is a graph illustratingthe quantification of bands in FIG. 35B. FIG. 35D is an image of animmunoblot illustrating levels of poly-ubiquitylated (poly-Ub) proteinsin the hippocampus in response to treatment with vehicle or IPAG.

FIGS. 36A-36C illustrate Sigma1 antagonist associated autophagy. FIG.36A, comprises representative images of translocation of GFP-tagged LC3(LC3-GFP) into autophagosomes in MDA-MB-468 (MDA468) breastadenocarcinoma cells. Only antagonist-treated MDA-MB-468 cells presentedLC3-GFP translocation to autophagosomes: 10 μM IPAG (antagonist) and 10μM PRE084 (agonist). Similar results were obtained with haloperidol(antagonist) and (+)SKF-10047 (agonist) (not shown). The images arerepresentative of at least 80 fields for each treatment condition. FIG.36B is an image of an immunoblot illustrating the finding that formationof GFP-LC3II the formation of GFP-LC3 positive punctae, an indicator ofautophagosome formation. Protein extracts from MDA-MB-468 cells stablytransfected with and expressing GFP-LC3, treated for 24 hours with 10 μMof the indicated Sigma1 ligand. FIG. 36C comprises images of immunoblotsillustrating that increased levels of the microtubule associated proteinlight chain 3BII isoform (LC3BII) is another broadly used indicator ofactivated autophagy. Treatment of a range of tumor cell lines with 10 μMIPAG for 24 hours resulted in a salient induction of LC3BII levels,whereas PRE084 did not significantly alter basal autophagy. Similarresults were obtained with haloperidol and (+)SKF-10047 (not shown).Legend: MCF-7 (breast adenocarcinoma), T47D (breast adenocarcinoma),DU145 (prostate adenocarcinoma), PC3 (prostate adenocarcinoma), HepG2(hepatocellular carcinoma), and Panc1 (pancreatic adenocarcinoma).

FIGS. 37A-37D illustrate that inhibition of autophagy accelerates and/orpotentiates Sigma1 antagonist induced apoptosis. FIG. 37A is a graphillustrating the time-course of Sigma antagonist-induced cell death asmeasured by flow cytometry. MDA-MB-468 cells were treated with DMSO(control), 10 μM IPAG (antagonist), or 10 μM PRE084 (agonist), fixed andstained with propidium iodide for DNA content analysis. The subG1population was quantified and used as a measure of cell death. Data arefrom 4 independent determinations. FIG. 37B is an image of a Westernblot of caspase 3 (Asp175) cleavage. IPAG induced apoptotic cell deathoccurred at the 48 hour treatment time point. FIG. 37C is a graphillustrating DA-MB-468 cells treated for 24 hours with a combination ofIPAG (10 μM) and autophagy inhibitor 3-methyladenine (3-MA, 5 mM). Celldeath was measured by trypan blue exclusion. FIG. 37D is an image of animmunoblot confirming apoptotic cell death by detection of cleavedcaspase 3 (Asp175) and cleaved PARP (Asp214). Cell death was observed at24 hours only when the Sigma antagonist and autophagy inhibitor werecombined. For FIGS. 37A-37C, statistical significance was determined bytwo-way ANOVA followed by Bonferroni post-test, ***P<0.001.

FIG. 38 is a series of graphs illustrating Sigma receptor antagonistpotentiates bortezomib-induced adenocarcinoma cell death. In vitro cellproliferation and cell death were quantified by Alamar blue reduction.To evaluate potentiation of proteasome inhibitor (bortezomib)-inducedcell death by IPAG, MDA-MB-468 cells were treated for 20 hours with a 10μM IPAG or 0.01 μM bortezomib alone or both drugs combined. Cell deathwas confirmed by trypan blue exclusion assay (data not shown). Data arerepresentative of an experiment performed in triplicate.

FIGS. 39A-39B illustrate that rapamycin modulates response to Sigma1antagonist mediated endoplasmic reticulum stress. MDA-MB-468 breastadenocarcinoma were treated for 20 hours with the indicatedconcentration of IPAG (Sigma1 antagonist) alone or in combination with0.1 μM Rapamycin. FIG. 39A is a series of images of immunoblotsevaluating levels of unfolded protein response (UPR) markers, ATF4 andBiP. FIG. 39B is a series of images of immunoblots evaluating levels ofphospho-JNK. This suggests that enhancement of mTOR-mediatedmacro-autophagy may mitigate Sigma1 antagonist induced ER stress.

FIGS. 40A-40C illustrate Sigma1 antagonist modulation of Akt, S6K, andERK phosphorylation. MDA-MB-468 breast tumor cells were treated for 24hours with 10 μM IPAG (Sigma1 antagonist) or (+)SKF-10047 (SKF, Sigma1agonist). Treated cells were harvested and cellular proteins wereextracted with a buffer containing 1% Sodium Dodecyl Sulfate (SDS) anddeoxycholate (DOC), supplemented with phosphatase and proteaseinhibitors. FIG. 40A is an image of an immunoblot illustrating Aktphosphorylation. Whereas Akt phosphorylation at serine 473 decreased3-fold in IPAG treated cells, (+)-SKF10047 treatment did not alter Aktphosphorylation. Akt phosphorylation at threonine 308 was not altered inany of the samples (not shown). Equivalent loading was confirmed with ananti-GAPDH antibody. FIG. 40B is an image of an immunoblot illustratingphosphorylation. Phosphorylation of mTOR substrate, p70S6K at threonine389 decreased 3-fold in IPAG, but not SKF treated cells. Total p70S6Kwas immunoblotted as a loading control. FIG. 40C is an immunoblotillustrating that p44/42 ERK1/2 phosphorylation at threonine 202 andtyrosine 204 was abrogated by 24 hour treatment with IPAG. Data shown isrepresentative of two independent experiments.

FIGS. 41A-41B illustrate work-flow schematics for the identification ofSigma1 associated proteins. FIG. 41A illustrates putative topology ofSigma1. A homobifunctional cross-linking agent, dithio-bis(succinimidylpropionate) (DSP), was used for intracellular cross-linking of Sigma1associated proteins to either of the two lysine residues of Sigma1.Amino acid residue E150 is indicated. FIG. 41B is a flow chartillustrating a dual affinity purification scheme to isolate and identifySigma1-HA-His6 associated proteins.

FIGS. 42A-42B illustrate Sigma1 associated proteins in a breast tumorcell line. FIG. 42A is a Western blot of dual affinity purifiedSigma1-HA-His6 sample, immunoblotted with a rabbit anti-hemagglutinin(HA) epitope, HRP conjugated antibody. The HA-agarose affinity columnwas made with a mouse monoclonal anti-HA antibody. FIG. 42B is a piechart illustrating organelle distribution of Sigma1 associated proteins.The identified proteins were not restricted to a single functionalcategory, nor were they exclusively located in indicated organelles. Innearly all cases, the Sigma1 associated proteins existed in multipleorganelles and in multiple cellular processes. LC/MS analysis subsequentto subcellular fractionation may provide more information concerningSigma1 associated protein interactions and responses to Sigma ligandtreatment. Legend: ER. endoplasmic reticulum; ERGIC, ER-Golgiintermediate compartment; PM, plasma membrane.

FIG. 43, comprising panels A-D, illustrates a proposed model for amechanism of Sigma1-mediated actions. Panel A is a model illustratingSigma1-HA-His6 quantal association. Panel B is a model illustratingquantal dissociation. Panel C is a model illustrating graded gain ofassociation. Panel D is a model illustrating graded loss of association.The Sigma1 “receptor” has no known intrinsic enzymatic or signalingfunction. Sigma1 may function as a novel molecular chaperone, and Sigma1antagonists may induce ER stress by altering its physical associationwith partner proteins. Putative Sigma1 ligand binding sites areindicated by triangles (Δ), which are distinct from IDR protein binding.

FIGS. 44A-44D illustrate the finding that p97/VCP is a Sigma1 associatedprotein. A novel assay was developed to evaluate Sigma1 ligandmodulation of Sigma1 functions involving protein-protein interactions.FIG. 44A is an image of an immunoblot confirming expression of p97/VCPand Sigma1-HA-His6 in detergent soluble cell extracts added to Ni-NTAresin (input). FIG. 44B is an image of an immunoblot relating to eluatefrom a Ni-NTA affinity purified fraction. Co-isolation/co-purificationof p97/VCP was accomplished with Sigma1-HA-His6, which wasisolated/purified by Ni-NTA. No p97/VCP was detected in an eluatefraction from Ni-NTA purified from protein extracts of non-transfected,parental cells that did not express Sigma1-HA-His6 (−). FIG. 44C is animage of an immunoblot confirming expression of p97/VCP andSigma1-HA-His6 in detergent-soluble cell extracts from MDA-MB-468 breastadenocarcinoma cells treated for 12 hours with 10 μM IPAG (Sigma1putative antagonist); the extracts were added to Ni-NTA resin (input).FIG. 44D is an image of an immunoblot illustrating eluate from a Ni-NTAaffinity purified fraction. Co-isolation/co-purification of p97/VCP wasaccomplished with Sigma1-HA-His6, which was isolated/purified by Ni-NTA.Lower levels of Sigma1-associated p97/VCP were detected in eluatefraction from Ni-NTA purified from protein extracts of MDA-MB-468 cellstreated for 12 hours with 10 μM IPAG (Sigma1 putative antagonist).Molecular mass is presented in kilodaltons (kDa).

FIGS. 45A-45B illustrate the finding that ubiquitylated proteins arebound to Sigma1. A novel assay was developed to evaluate Sigma1 ligandmodulation of Sigma1 functions involving association with ubiquitylatedproteins. FIG. 45A is an image of an immunoblot confirming expression ofSigma1-HA-His6 and induction of ubiquitylated protein levels indetergent soluble cell extracts added to Ni-NTA resin (input) fromMDA-MB-468 breast adenocarcinoma cells treated for 12 hours with 10 μMIPAG (Sigma1 putative antagonist). FIG. 45B is an image of an immunoblotof eluate from a Ni-NTA affinity purified fraction.Co-isolation/co-purification of poly-ubiquitylated proteins withSigma1-HA-His6 were isolated/purified by Ni-NTA. Increased levels ofSigma1-associated ubiquitylated proteins were detected in an eluatefraction from Ni-NTA purified from protein extracts of MDA-MB-468 cellstreated for 12 hours with 10 μM IPAG (Sigma1 putative antagonist).Molecular mass is presented in kilodaltons (kDa).

FIGS. 46A-46G illustrate bioinformatics prediction and mutationalanalysis of an intrinsically disordered region in the Sigma1 cytoplasmictail. FIG. 46A is a schematic illustration of Sigma1 membrane topology.The amino acid sequence surrounding residue 150 is shown at the bottom.This sequence also corresponds to synthetic blocking peptides that havebeen generated against this region. FIG. 46B is an image of a denaturingSDS-PAGE gel of WT and mutant Sigma1 from HEK293T transient transfectantcell lysates. Apparent molecular mass (M_(r)) is presented inkilodaltons (kDa). FIG. 46C is a native PFO-PAGE of same cell lysates.FIG. 46D is an illustration of conformational differences between wildtype and mutant Sigma1 predicted in FIG. 46A. and revealed by SDS-PAGE.FIG. 46E is an illustration of conformational differences between wildtype and mutant Sigma1 predicted in FIG. 46A and revealed by PFO-PAGE.FIG. 46F is a graph illustrating that intrinsic disorder in the Sigma1using PONDR VL-XT (URL and reference) indicates a short intrinsicallydisordered region (IDR) within the murine Sigma1 carboxy-terminal tail,corresponding to residues underlined in FIG. 46A. The black downwardarrow points to disordered region. FIG. 46G is a graph illustrating thefinding that E150A mutation resulted in loss of intrinsic disorder.

FIG. 47 illustrates the UVCD spectral analysis of Sigma1 C-tail peptide.The intrinsically disordered region in the Sigma1 cytoplasmic tail wasexamined through the temperature-dependent ultraviolet circulardichroism (UVCD) spectra of (a) the native Sigma1 peptide residues137-159 and (b) the corresponding mutant Sigma1 peptide (E150A). Thearrows indicate the increasing temperature from 283-363K. Panels (c) and(d) show the negative maximal dichroism (Δε_(193 nm)) obtained from theUVCD spectra plotted as a function of temperature for the native andmutant peptide, respectively.

FIGS. 48A-48B illustrate the finding that the Sigma1 IDR mutantabrogates ligand mediated induction of ER stress, and can be used as atool to evaluate aspects of Sigma1 ligand mediated ER stress response.HEK293T cells were stably transfected with Sigma1-HA-His₆ (WT^(OE)) orSigma1E150A-HA-His₆ (E150A^(OE)) and treated for ˜16 hours with DMSO or10 μM IPAG (Sigma1 “antagonist”). FIG. 48A is a series of images ofimmunoblots of GRP78/BiP (BiP), a marker of unfolded protein responseand endoplasmic reticulum (ER) stress. FIG. 48B is a graph illustratingthe quantification of BiP induction from three determinations.

FIGS. 49A-49D illustrate the finding that a point mutation in Sigma1cytoplasmic tail inhibited the response to Sigma1 antagonists mediatedcell cycle arrest, and may be used as a novel tool to evaluate Sigmaligand modulation of cell cycle and proliferation. Treatment with aSigma1 antagonist resulted in accumulation of cells in G1 phase of thecell cycle. FIG. 49A is a graph illustrating the percentage of G1, S,and G2 phase cells in T47D cells treated for 24 hours with DMSO (vehiclecontrol), 1 μM IPAG (Sigma1 antagonist), or 10 μM (+)-SKF-10047 (Sigma1agonist). FIG. 49B is a graph illustrating the percentage of G1, S, andG2 phase cells in MDA-MB-468 cells treated for 24 hours with DMSO(vehicle control), 10 μM IPAG (Sigma1 antagonist), or 10 μM (+)SKF-10047(Sigma1 agonist). In contrast to IPAG treatment, control (DMSO) and(+)SKF10047 (SKF) treated cells did not present significant differencein cell cycle profile. FIG. 49C is a graph illustrating ligand-mediatedchanges to cell proliferation measured by MTT assay. Proliferation ofparental, wild-type, Sigma1 expressing cells was measured in standardcell culture medium and compared to proliferation in medium (solid blackline) containing 10 μM BD1047 (Sigma1 antagonist, broken black line).MTT assay was performed at the indicated time points (treatment days).FIG. 49D is a graph illustrating proliferation of E150A mutant (alE150A)expressing cells measured in standard cell culture medium and comparedto proliferation in medium (solid line) containing 10 μM BD1047 (Sigma1antagonist, broken line).

FIGS. 50A-50B illustrate the finding that the binding of Sigma1 smallmolecule ligands were not altered by cytoplasmic tail mutation, and maybe used as a tool to evaluate post-ligand-binding steps mediated bySigma1 ligand-receptor interactions. FIG. 50A is a graph illustrating[³H]-(+)-pentazocine (selective Sigma1 ligand) binding saturation. FIG.50B illustrates the results of [³H]-(+)-pentazocine competition binding.Binding was performed with [³H]-(+)-pentazocine (3 nM) in HEK cellmembranes from Sigma1-HA transfected cells (clone 320-3-3), Sigma1mutated (E150A-HA) cells (clone 167-11-7) and Sigma1 mutated (E150K-HA)cells (clone 320-32-12). Despite the profound effect of the mutation onthe inhibition of cell proliferation, the E150A mutation did not alter3H-Sigma ligand binding.

FIG. 51 is an immunoblot illustrating the finding that Sigma1 ligandtreatment is associated with ER stress. MDA-MB-468 breast adenocarcinomacells were treated for ˜16 hours with DMSO control, IPAG (10 μM),haloperidol (10 μM), PB28 (20 μM), rimcazole (20 μM), PRE084 (20 μM),(+)SKF10047 [(+)SKF, (20 μM)], or (+)pentazocine [(+)PTZ, 20[μM].Detergent-soluble total cell extracts were resolved by 10% SDS-PAGE andimmunoblotted to detect levels of GRP78/BiP (BiP), Sigma1, and βactin(loading control). Putative Sigma1 “antagonist” and “agonist” areindicated.

FIGS. 52A-52B illustrate 3-(4-fluorophenoxy) propan-1-amine, which canact as a haloperidol amine surrogate. FIG. 52A is a scheme illustratingthe synthesis of 3-(4-fluorophenoxy)propan-1-amine, beginning with1-(3-chloropropoxy)-4-fluorobenzene. FIG. 52B illustrates the ¹H NMRspectrum of 3-(4-fluorophenoxy)propan-1-amine.

FIG. 53 is a synthetic scheme illustrating a general synthetic strategytoward the synthesis of N-(4-iodophenyl)cyanamide.

FIGS. 54A-54D illustrate a synthetic strategy toward guanidines. FIG.54A is a scheme illustrating a general synthesis of guanidines. FIG. 54Bis the ¹H NMR spectrum illustrating formation of symmetric andnon-symmetric 4-iodophenyl guanidines (structures are pictured below thespectrum). FIG. 54C is the HPLC trace for the mixture of non-symmetricguanidine and dimer. FIG. 54D is the MS trace for the mixture ofnon-symmetric guanidine and dimer.

FIGS. 55A-55C illustrate the HPLC purification of 1-3-(4-fluorophenoxy)propyl)-3-(4-iodophenyl)guanidine for testing. FIG. 55A illustrates theHPLC trace of the purified compound. FIG. 55B is a trace of the HPLCfractions. The pure fraction is highlighted (black arrow). FIG. 55C isthe structure of 1-3-(4-fluorophenoxy)propyl)-3-(4-iodophenyl)guanidine.

FIGS. 56A-56D illustrate the finding that the novel Sigma1 ligand,JMS-51-58 [1-3-(4-fluorophenoxy)propyl)-3-(4-iodophenyl)guanidine],induced autophagy. FIG. 56A is an immunoblot illustrating MDA-MB-468breast adenocarcinoma cells stably transfected with GFP-LC3 (greenfluorescent protein tagged light chain 3, an autophagy marker) treatedfor ˜16 hours with 10 μM IPAG or 10 μM 1-3 JMS-51-58. The appearance ofGFP-LC3II was an indication of autophagosome formation. FIG. 56B is animage illustrating formation of autophagosomes (GFP-LC3 punctae) inMDA468 (GFP-LC3) treated with 10 μM JMS-51-58 as in FIG. 56A. The arrowpoints to an example of GFP-LC3-positive autophagosomes. The white barat the bottom of the image indicates 100 μm. FIG. 56C is an image of animmunoblot illustrating the appearance of endogenous LC3II, a marker ofautophagosome formation, in MDA-MB-468 breast adenocarcinoma cellstreated for 16 hours with 10 μM of JMS-51-58. FIG. 56D is an image of animmunoblot illustrating the appearance of endogenous LC3II, a marker ofautophagosome formation, in T47D breast adenocarcinoma cells treated for16 hours with 10 μM JMS-51-58.

FIGS. 57A-57B illustrate the finding that the novel Sigma1 ligand,JMS-51-58, mediated translational arrest. FIG. 57A is an image of animmunoblot illustrating MDA-MB-468 breast adenocarcinoma cells treatedfor 16 hours with 10 μM of JMS-51-58. FIG. 57B is an image of animmunoblot illustrating T47D breast adenocarcinoma cells treated for 16hours with 10 μM of JMS-51-58. The levels of phosphothreonine389-p70S6Kinase (P-S6K) and phosphoserine 65-4E-BP1 (P-4E-BP1) wereevaluated by immunoblot. Decreased levels of both phosphoprotein levelsindicated diminished protein synthesis.

FIGS. 58A-58B illustrate the finding that the novel Sigma1 ligandJMS-51-58 mediated ER stress response. FIG. 58A is an image of animmunoblot of MDA-MB-468 breast adenocarcinoma cells treated for 16hours with 10 μM JMS-51-58. FIG. 58B is an image of an immunoblot ofT47D breast adenocarcinoma cells treated for 16 hours with 10 μMJMS-51-58. The levels of phospho-Thr 180/Tyr 182 p38MAPK (P-p38MAPK) andGRP78/BiP and were evaluated by immunoblot.

FIGS. 59A-59B illustrate the finding that the novel Sigma1 ligandJMS-51-58 modulated the ubiquitin proteasome system. FIG. 59A is animage of an immunoblot evaluating the levels of poly-ubiquitylatedproteins of MDA-MB-468 breast adenocarcinoma treated for 16 hours with10 μM of JMS-51-58. FIG. 59B is an image of an immunoblot evaluating thelevels of poly-ubiquitylated proteins of MDA-MB-231 breastadenocarcinoma cells treated for 16 hours with 10 μM of JMS-51-58. Thelevels of poly-ubiquitylated proteins were evaluated by immunoblot.JMS-51-58 treatment resulted in increased ubiquitylated protein levelscompared to the DMSO control.

FIGS. 60A-60D illustrate the finding that the novel Sigma1 ligandJMS-51-58 inhibited tumor cell proliferation. In vitro cellproliferation was quantified by Alamar blue assay. Theanti-proliferative effects of four drug concentrations of novel Sigma1small molecule ligand JMS-51-2^(nd)-58 were quantified after 40-70 hoursof treatment. Data are representative of an experiment formed intriplicate for each cell line. FIG. 60A is a graph illustrating celldeath in MDA-MB-468 breast adenocarcinoma cell cultures. FIG. 60B is agraph illustrating cell death in T47D breast adenocarcinoma cellcultures. FIG. 60C is a graph illustrating cell death in MDA-MB-231breast adenocarcinoma cell cultures. FIG. 60D is a graph illustratingcell death in PC3 prostate adenocarcinoma cell cultures.

FIGS. 61A-61B are a set of graphs illustrating the finding that thenovel Sigma1 ligand JMS-51-58 potentiated proteasome inhibitor mediatedinhibition of tumor cell proliferation. In vitro cell proliferation wasquantified by Alamar blue assay. The anti-proliferative effects of fourdrug concentrations of novel Sigma1 small molecule ligand JMS-51-58,were quantified after ˜70 hours of treatment of MDA-MB-231 breastadenocarcinoma cell cultures. FIG. 61A is a graph illustrating that inMDA-MB-231 cell culture, 3 μM of JMS-51-58 did not alter cellproliferation or rates of cell death. FIG. 61B is a graph illustratingthat the sub-lethal dose of JMS-51-58 potentiated the cell proliferationinhibiting effects of bortezomib (BTZ). Note the shift to the left ofthe dose-response curve (broken line). Data are representative of anexperiment performed in triplicate.

FIGS. 62A-62B are a set of graphs illustrating the finding that thenovel Sigma1 ligand JMS-51-58 potentiated proteasome inhibitor mediatedtumor cell death. In vitro cell death was quantified by Trypan blueexclusion assay. To evaluate potentiation of proteasome inhibitor(bortezomib)-induced cell death by JMS-51-58, MDA-MB-468 cells weretreated for 20 hours with JMS-51-58 (10 μM) alone, bortezomib (1 nM, 10nM, 100 nM) alone or both drugs combined. Treatment with IPAG (10 μM)alone or combined with bortezomib (1 nM, 10 nM, 100 nM) was performed inparallel. Data are representative of an experiment performed intriplicate.

FIGS. 63A-63D illustrate the finding that the novel Sigma1 ligandJMS-51-58 inhibited adhesion of metastatic breast and prostateadenocarcinoma cell lines, revealing its potential for use as aninhibitor of tumor cell metastasis. In vitro cell adhesion wasquantified by Alamar blue assay. MDA-MB-231 and PC3 cells were detachedwith 0.25% trypsin+2 mM EDTA and reseeded onto 96 well plates. At thetime of cell seeding, IPAG (Sigma1 putative antagonist) was added at theindicated doses to wells containing adenocarcinoma cells. The suspendedcells were allowed to sediment and adhere for 16 hours in the presenceof drug. Subsequently, the culture medium was removed by aspiration,each well washed once with 0.2 ml PBS (without calcium and magnesium),and Alamar blue assay was performed to quantify the number of live cellsadhered to the surface of each well. FIG. 63A is a graph illustratingcell adherence in MDA-MB-231 breast adenocarcinoma cells. FIG. 63B is agraph illustrating cell adherence in PC3 prostate adenocarcinoma cells.The same procedure was performed using the indicated concentrations ofJMS-51-58. FIG. 63C is a graph illustrating cell adherence in MDA-MB-231breast adenocarcinoma cells. FIG. 63D is a graph illustrating celladherence in PC3 prostate adenocarcinoma cells. Standard deviation barsare shown.

FIGS. 64A-64C illustrate the incorporation of fluorophores into Sigmaligands for use as novel fluorescent probes to study biologicalphenomena in living cells. FIG. 64A is the structure of 7-amino-4-methylcoumarin, a fluorescent dye. FIG. 64B is the structure of a fluorescentprobe (a Sigma1 ligand comprising a fluorophore) useful to study Sigmareceptor biology in living cells. FIG. 64C is a scheme illustrating thesynthesis of the fluorescent probe.

FIG. 65 illustrates the modulation of androgen receptor levels inprostate cancer by a Sigma1 antagonist. Androgen receptor (AR) positiveprostate adenocarcinoma cell line, LNCaP, was treated with increasingdoses (1-10 μM) of Sigma1 antagonist, IPAG, for 16 hours andsubsequently cells were harvested, and proteins were extracted andimmunoblotted to evaluate Sigma1 ligand mediated changes in AR proteinlevels, changes in protein levels AR responsive genes (Cyclin D1, p21),induction of UPR (band migration shift of PERK, increased levels ofBiP), induction of autophagy (appearance of LC3II, increased levels ofp62SQSTM), changes in protein synthesis (in this case, absence ofchanges in protein synthesis marker phospho-4EBP1), and increased levelsof ubiquitylated proteins. βactin served as a loading control.

FIGS. 66A-66H illustrate the non-limiting mechanism(s) of Sigma1 ligandactions, wherein Sigma ligand treatment induces differentiallocalization of Sigma1. FIG. 66A is an immunoblot that illustrates thatSigma1 steady-state protein levels do not change in response to IPAGtreatment. FIG. 66B is an immunoblot of the fractions collected frombiochemical sub-cellular fractionation assay. This immunoblotdemonstrates that Sigma1 distribution is altered in response to IPAGtreatment. FIG. 66C is a graph illustrating quantification of bands inFIG. 66B. FIG. 66D is a set of images of confocal immunofluorescencemicroscopy of Sigma1 in SKBR3 cells treated for 12-16 hours with IPAG.Green signal indicated Sigma1, blue signal (DAPI) indicated nucleus.FIG. 66E is a set of graphs illustrating quantification of fractionscollected from biochemical sub-cellular fractionation assay performedusing SKBR3 cells. Red line represents fractionation following 12-16hour treatment with IPAG, the blue line represents fractionation of12-16 hour DMSO (vehicle (control) treated cells. Graph (i):Quantification of VCP fractionation immunoblot. Graph (ii):Quantification of p62SQSTM fractionation immunoblot. FIG. 66F is a setof confocal microscopy images, with evidence of co-localization ofp97/VCP (VCP) and autophagosome marker GFP-LC3II (LC3). This confocalimage demonstrated presence of VCP in autophagosomes (indicated by whitearrows in merged image, lower right panel). FIG. 66G is a set ofconfocal microscopy images, with evidence of co-localization of p62SQSTMand autophagosome marker GFP-LC3II (LC3). This confocal imagedemonstrates presence of p62SQSTM in autophagosomes (indicated by whitearrows in merged image, lower right panel). p62SQSTM is an adaptorprotein for the transport and incorporation of ubiquitylated proteinsinto autophagosomes. Functional and physical association with LC3II andfunctional interaction with VCP are reported in the literature. FIG. 66His a set of confocal microscopy images, with evidence of co-localizationof poly-ubiquitylated proteins and p62SQSTM. This confocal imagedemonstrated presence of poly-ubiquitylated proteins in autophagosomesthat contain p⁶²SQSTM. The ubiquitin-p62SQSTM co-localization anddistribution pattern changed subsequent to IPAG treatment. The dataillustrated here support the finding that treatment of a cell with an ERstress inducing Sigma ligand (e.g., IPAG) did not alter totalSigmalreceptor levels, but did alter the subcellular localization ofSigma1, with Sigma1 redistributed and concentrated in the ER. Sigma1functional related and physical associated proteins were redistributedto the other parts of the cell, especially into autophagosome. Further,prolonged ER stress induced by IPAG treatment caused cancer cell death.This is consistent with data presented in FIGS. 43-44, wherein changesin protein associations were observed in response to Sigma ligandtreatment.

FIGS. 67A-67F illustrate the finding that Sigma1 ligands may be used tomodulate (in this case, decrease) the levels of growth factor receptorssuch as HER2 and HER3 in breast cancer cells, in the absence ofdetectable cell death. IPAG time and dose-dependently causes decreasedlevels of HER2/3 in SkBr-3 cells. Short exposure to IPAG or lower dosageinduces ubiquitin-mediated autophagosomal degradation of HER2/3.Prolonged IPAG treatment or higher dosage caused translational arrest.At later stage, a combined effect of autophagosomal degradation andtranslational arrest significantly reduced HER2/3 level in SkBr-3 cells.FIG. 67A is a set of immunoblots of HER2 following treatment with IPAG.Detergent soluble whole-cell lysates were solved by denaturing SDS-PAGE.(i) 1-to-24 hour (h) treatment with 10 μM IPAG or 1 μM thapsigargin(TG). (ii) Following 12 hour treatment with 1, 3, 10, 20 μM IPAG. FIG.67B is a set of immunoblots of HER3 following treatment with IPAG.Detergent soluble whole-cell lysates were solved by denaturing SDS-PAGE.(i) 1-to-24 hour (h) treatment with 10 μM IPAG or 1 μM thapsigargin(TG). (ii) Following 12 hour treatment with 1, 3, 10, 20 μM IPAG. FIG.67C is a set of immunoblots of detergent soluble whole-cell lysatessolved by denaturing SDS-PAGE. (i) Immunoblot of translationalregulation, autophagy, and protein ubiquitylation markers following1-to-24 hour (h) treatment with 10 μM IPAG. (ii) Immunoblot to evaluatedrug dose-response—12 hour treatment with 1, 3, 10, 20 μM IPAG—using thesame markers indicated in (i). FIG. 67D is a series of immunoblot of thefractions collected from biochemical sub-cellular fractionation assayperformed using SKBR3 cells. (i) This immunoblot demonstrates that theintracellular distribution of ubiquitylated proteins is altered inresponse to IPAG treatment. Note salient shift in band intensity frompredominantly fraction 4-6 in control (DMSO) treated cells compared tofraction 3-5 in IPAG treated cells. (ii) Fractions in whichautophagosome marker LC3II is found correlates with ubiquitylatedproteins in (i). (iii) Quantification of bands in (i). FIG. 67E is aseries of confocal microscopy images, with evidence of co-localizationof HER2 and ubiquitin (Ub). This confocal image demonstrated decreasedlevels of HER2 in response to IPAG treatment (intensity of green signalin DMSO upper right compared to IPAG upper right panel) and concentratedco-localization of Ub and HER2 subsequent to IPAG treatment (indicatedby white arrows in merged image, lower right panel). FIG. 67Fillustrates biochemical subcellular fractionation. (i) Immunoblot ofHER3 in cell lysate fractions from SKBR3 following 16 hour (h) treatmentwith 10 μM IPAG. (ii) Quantification of HER3 band in DMSO (vehiclecontrol) treated conditions. (iii) Quantification of HER3 band in IPAGtreated conditions.

FIG. 68 is a series of confocal microscopy image illustrating theevidence of co-localization of poly-ubiquitylated proteins andautophagosome marker GFP-LC3II (LC3). This confocal image demonstratespresence of poly-ubiquitylated proteins in autophagosomes (indicated bywhite arrows in merged image, lower right panel).

FIGS. 69A-69B illustrats the finding that Sigma1 ligand, JMS-57-10,mediates UPR, modulates protein translation, and autophagy in theabsence of cell death. (FIG. 69A) Alamar blue assay used to quantify therelative number of viable cells (live cells) following 72 hours oftreatment with a range of doses of the indicated Sigma1 ligands. Redcircle highlights effects of treatment with JMS-57-10 (57-10). Note thatfor all three cell lines, JMS-57-10 did not elicit appreciable celldeath under these conditions. Therefore, the decrease in the relative %of live cells observed for T47D represents inhibited cell proliferation.(FIG. 69B) Immunoblot to evaluate induction of UPR, translationalregulation, and autophagy in response to IPAG and JMS-57-10 (57-10).

FIG. 70 is a series of confocal microscopy images illustrating thefinding that Sigma1 is present in predominantly in the ER of SKBR3breast cancer cells. Confocal immunofluorescence microscopy demonstratedco-localization of Sigma1 and calnexin, a resident endoplasmic reticulum(ER) protein. The result indicated that Sigma1 remained ER localized,albeit with apparently different distribution pattern, in cells treatedfor 12-16 hours with IPAG. Yellow signal (merge) indicatedco-localization.

FIG. 71 is a synthetic scheme illustrating the synthesis of2-(4-(4-methyl-2-oxo-2H-chromen-7-yl)piperazin-1-yl)ethyl1-phenylcyclohexanecarboxylate, a fluorescent probe useful in theinvention.

FIG. 72 is a synthetic scheme illustrating the synthesis of1-(4-methoxyphenyl)-3-(3-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)propyl)guanidineand1-(4-iodophenyl)-3-(3-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)propyl)guanidine,fluorescent probes useful in the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the unexpected discovery of novelcompounds that bind to and modulate the activity of the Sigma receptor.These compounds are useful in the treatment of Sigma receptor-relateddiseases and disorders, either alone or in combination with at least oneadditional therapeutic agent. In one embodiment, the Sigma modulator ofthe invention is a Sigma antagonist, inverse agonist or agonist. Inanother embodiment, the Sigma modulator of the invention is a Sigmaantagonist. In yet another embodiment, the Sigma receptor is a Sigma1receptor (also known as Sigma1).

The present invention includes novel methods of treating, amelioratingor preventing a Sigma receptor-related disease or disorder using thecompounds of the invention. In one embodiment, the Sigmareceptor-related disease or disorder is selected from the groupcomprising cancer, neuropathic pain, depression, substance abuse,epilepsy, psychosis, Alzheimer's disease, Parkinson's disease,frontotemporal lobar degeneration (FTLD), amyotrophic lateral sclerosis(ALS) and combinations thereof. In another embodiment, the cancer isselected from the group consisting of prostate cancer, liver cancer,pancreas cancer, CNS tumors (including brain tumors), breast cancer,neuroblastoma, leukemia, and combinations thereof.

The present invention also includes novel methods of treating,ameliorating or preventing a Sigma receptor-related disease or disorderusing the compounds of the invention in combination with therapeuticagents that target the UPR and/or autophagic survival pathways. In apreferred embodiment, the Sigma receptor-related disease or disorder iscancer.

Compounds useful within the methods of the invention include thecompounds of Formula (I) and Formula (II) as described elsewhere herein,as well as any compound known to be a Sigma antagonist, agonist orinverse agonist, such as but not limited to haloperidol, IPAG, PB28,rimcazole, BD1063, BD1047, PRE084, NE100, (+)-SKF10047, (+)-pentazocine,and any combinations thereof. The invention contemplates using any ofthese compounds to modulate cellular protein synthesis, processing,and/or degradation in a subject in need thereof.

In one aspect, the compounds of the invention are useful in thetreatment of cancers and neurodegenerative disorders wherein cellularfunctions can be selectively targeted by Sigma ligands.

As illustrated herein (FIGS. 69A-69B), a compound of the invention wasshown not to be cytotoxic to several tumor cell lines tested. Despitethis lack of cytotoxicity, the compound still induced UPR. The compoundsof the invention may thus be used to treat a neurodegenerative diseaseand other pathologies and disorders wherein modulation of proteinhomeostasis (such as synthesis, folding, processing, or degradation ofproteins) could be beneficial.

As illustrated in FIGS. 66A-66H, treatment of a cell with an ER stressinducing Sigma ligand (e.g., IPAG) did not alter Sigma1 levels, but didalter the subcellular localization of Sigma1. Consistently, asillustrated herein (FIGS. 43 and 44A-44D), changes in proteinassociations were observed in response to Sigma ligand treatment. In anembodiment, ligand mediated changes to Sigma1 partner proteinassociations and corresponding/consequent changes in Sigma1 subcellularlocalization may provide the basis for biochemical mechanism studies toestablish Sigma ligand structure-activity-relationships.

As illustrated in FIGS. 67A-67F, Sigma1 ligands modulate (in this case,decrease) the levels of growth factor receptors such as HER2 and HER3 inbreast cancer cells, in the absence of detectable cell death. In oneembodiment, a compound of the invention may be used to treat cancer in asubject in need thereof, wherein administering of the compound to thesubject causes degradation of a growth factor receptor in the tumorcells. In another embodiment, the growth factor receptor comprises EGFR(epidermal growth factor receptor), HER2, HER3, p95HER2 (truncated formof HER2), androgen receptor, or any combinations thereof. In yet anotherembodiment, the cancer comprises breast cancer or prostate cancer. Inyet another embodiment, the prostate cancer comprisescastration-sensitive or castration-insensitive prostate cancer. Sigmaligands may thus be used as selective cytotoxics or selective inhibitorsof cell growth without cytotoxicity. This broadens the utility of Sigmaligands beyond simple cytotoxic agents, and it demonstrates thepotential versatility of the compounds of the invention.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described.

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of +20% or +10%, more preferably +5%, even more preferably+1%, and still more preferably +0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

The term “abnormal,” when used in the context of organisms, tissues,cells or components thereof, refers to those organisms, tissues, cellsor components thereof that differ in at least one observable ordetectable characteristic (e.g., age, treatment, time of day, etc.) fromthose organisms, tissues, cells or components thereof that display the“normal” (expected) respective characteristic. Characteristics that arenormal or expected for one cell or tissue type might be abnormal for adifferent cell or tissue type.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate.

In contrast, a “disorder” in an animal is a state of health in which theanimal is able to maintain homeostasis, but in which the animal's stateof health is less favorable than it would be in the absence of thedisorder. Left untreated, a disorder does not necessarily cause afurther decrease in the animal's state of health.

A disease or disorder is “alleviated” if the severity of a symptom ofthe disease or disorder, the frequency with which such a symptom isexperienced by a patient, or both, is reduced.

As used herein, the term “Sigma” refers to the Sigma1 receptor (Sigma1),Sigma2 receptor (Sigma2), any splice variant thereof or any isoformthereof.

As used herein, a “Sigma receptor modulator” is a compound that binds tothe Sigma receptor and modifies the activity or biological function ofthe receptor as compared to the activity or biological function of thereceptor in the absence of the modulator. The modulator may be areceptor agonist, which is able to activate the receptor and cause abiological response that is enhanced over the baseline activity of theunbound receptor. The modulator may be a partial agonist, which does notactivate the receptor thoroughly and causes a biological response thatis smaller in magnitude compared to those of full agonists. Themodulator may be a receptor antagonist, which binds to the receptor butdoes not activate it, resulting in receptor blockage and inhibiting thebinding of other agonists. An antagonist does not diminish the baselineintracellular response in the absence of an agonist. The modulator maybe an inverse agonistic, which reduces the activity of the receptor byinhibiting its constitutive activity.

The terms “patient,” “subject,” “individual,” and the like are usedinterchangeably herein, and refer to any animal, or cells thereofwhether in vitro or in situ, amenable to the methods described herein.In a non-limiting embodiment, the patient, subject or individual is ahuman.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs of pathology, for the purpose of diminishing oreliminating those signs.

As used herein, the term “treatment” or “treating” is defined as theapplication or administration of a therapeutic agent, i.e., a compoundof the invention (alone or in combination with another pharmaceuticalagent), to a patient, or application or administration of a therapeuticagent to an isolated tissue or cell line from a patient (e.g., fordiagnosis or ex vivo applications), who has a condition contemplatedherein, a symptom of a condition contemplated herein or the potential todevelop a condition contemplated herein, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve or affect acondition contemplated herein, the symptoms of a condition contemplatedherein or the potential to develop a condition contemplated herein. Suchtreatments may be specifically tailored or modified, based on knowledgeobtained from the field of pharmacogenomics.

As used herein, the term “composition” or “pharmaceutical composition”refers to a mixture of at least one compound useful within the inventionwith a pharmaceutically acceptable carrier. The pharmaceuticalcomposition facilitates administration of the compound to a patient orsubject. Multiple techniques of administering a compound exist in theart including, but not limited to, intravenous, oral, aerosol,parenteral, ophthalmic, pulmonary and topical administration.

The phrase “therapeutically effective amount,” as used herein, refers toan amount that is sufficient or effective to prevent or treat (delay orprevent the onset of, prevent the progression of, inhibit, decrease orreverse) a disease or condition associated with the Sigma receptor,including alleviating symptoms of such diseases.

As used herein, the terms “effective amount,” “pharmaceuticallyeffective amount” and “therapeutically effective amount” refer to anontoxic but sufficient amount of an agent to provide the desiredbiological result. That result may be reduction and/or alleviation ofthe signs, symptoms, or causes of a disease, or any other desiredalteration of a biological system. An appropriate therapeutic amount inany individual case may be determined by one of ordinary skill in theart using routine experimentation.

An “effective amount” of a delivery vehicle is that amount sufficient toeffectively bind or deliver a compound.

As used herein, the term “potency” refers to the dose needed to producehalf the maximal response (ED₅₀).

As used herein, the term “efficacy” refers to the maximal effect(E_(max)) achieved within an assay.

As used herein, the term “PRE084” refers to 2-morpholin-4-ylethyl1-phenylcyclohexane-1-carboxylate or a salt thereof.

As used herein, the term “BD1047” refers toN′-[2-(3,4-dichlorophenyl)ethyl]-N,N,N′-trimethylethane-1,2-diamine or asalt thereof.

As used herein, the term “BD1063” refers to1-[2-(3,4-dichlorophenyl)ethyl]-4-methylpiperazine or a salt thereof.

As used herein, the term “haloperidol” refers to4-[4-(4-chlorophenyl)-4-hydroxy-1-piperidyl]-1-(4-fluorophenyl)-butan-1-oneor a salt thereof.

As used herein, the term “(+)-SKF10047” refers to [2S-(2a,6a,11R*]-1,2,3,4,5,6-hexahydro-6,11-dimethyl-3-(2-propenyl)-2,6-methano-3-benzazocin-8-olor a salt thereof.

As used herein, the term “(+)-pentazocine” refers to(+)-[2S-(2,6,11R*)]-1,2,3,4,5,6-hexahydro-6,11-dimethyl-3-(3-methyl-2-butenyl)-2,6-methano-3-benzazocin-8-olor a salt thereof.

As used herein, the term “rimcazole” refers to9-{3-[(3R,5S)-3,5-dimethylpiperazin-1-yl]propyl}-9H-carbazole or a saltthereof.

As used herein, the term “PB28” refers to1-cyclohexyl-4-[3-(5-methoxy-1,2,3,4-tetra-hydronaphthalen-1-yl)propyl]piperazineor a salt thereof.

As used herein, the term “IPAG” refers to1-(4-iodophenyl)-3-(2-adamantyl)guanidine or a salt thereof.

As used herein, the term “NE100” refers to4-methoxy-3-(2-phenylethoxy)-N,N-dipropylbenzeneethanamine hydrochlorideor a salt thereof.

As used herein, the term “E64d” refers to (2S,3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester or asalt thereof.

As used herein, the term “methyladenine” refers to 3-methyladenine or asalt thereof.

As used herein, the term “tamoxifen” refers to(Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethylethanamine or asalt thereof.

As used herein, the term “pharmaceutically acceptable” refers to amaterial, such as a carrier or diluent, which does not abrogate thebiological activity or properties of the compound, and is relativelynon-toxic, i.e., the material may be administered to an individualwithout causing undesirable biological effects or interacting in adeleterious manner with any of the components of the composition inwhich it is contained.

As used herein, the language “pharmaceutically acceptable salt” refersto a salt of the administered compounds prepared from pharmaceuticallyacceptable non-toxic acids, including inorganic acids, organic acids,solvates, hydrates, or clathrates thereof. Examples of such inorganicacids are hydrochloric, hydrobromic, hydroiodic, nitric, sulfuric,phosphoric, acetic, hexafluorophosphoric, citric, gluconic, benzoic,propionic, butyric, sulfosalicylic, maleic, lauric, malic, fumaric,succinic, tartaric, amsonic, pamoic, p-tolunenesulfonic, and mesylic.Appropriate organic acids may be selected, for example, from aliphatic,aromatic, carboxylic and sulfonic classes of organic acids, examples ofwhich are formic, acetic, propionic, succinic, camphorsulfonic, citric,fumaric, gluconic, isethionic, lactic, malic, mucic, tartaric,para-toluenesulfonic, glycolic, glucuronic, maleic, furoic, glutamic,benzoic, anthranilic, salicylic, phenylacetic, mandelic, embonic(pamoic), methanesulfonic, ethanesulfonic, pantothenic, benzenesulfonic(besylate), stearic, sulfanilic, alginic, galacturonic, and the like.Furthermore, pharmaceutically acceptable salts include, by way ofnon-limiting example, alkaline earth metal salts (e.g., calcium ormagnesium), alkali metal salts (e.g., sodium-dependent or potassium),and ammonium salts.

As used herein, the term “pharmaceutically acceptable carrier” means apharmaceutically acceptable material, composition or carrier, such as aliquid or solid filler, stabilizer, dispersing agent, suspending agent,diluent, excipient, thickening agent, solvent or encapsulating material,involved in carrying or transporting a compound useful within theinvention within or to the patient such that it may perform its intendedfunction. Typically, such constructs are carried or transported from oneorgan, or portion of the body, to another organ, or portion of the body.Each carrier must be “acceptable” in the sense of being compatible withthe other ingredients of the formulation, including the compound usefulwithin the invention, and not injurious to the patient. Some examples ofmaterials that may serve as pharmaceutically acceptable carriersinclude: sugars, such as lactose, glucose and sucrose; starches, such ascorn starch and potato starch; cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;powdered tragacanth; malt; gelatin; talc; excipients, such as cocoabutter and suppository waxes; oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols,such as propylene glycol; polyols, such as glycerin, sorbitol, mannitoland polyethylene glycol; esters, such as ethyl oleate and ethyl laurate;agar; buffering agents, such as magnesium hydroxide and aluminumhydroxide; surface active agents; alginic acid; pyrogen-free water;isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffersolutions; and other non-toxic compatible substances employed inpharmaceutical formulations. As used herein, “pharmaceuticallyacceptable carrier” also includes any and all coatings, antibacterialand antifungal agents, and absorption delaying agents, and the like thatare compatible with the activity of the compound useful within theinvention, and are physiologically acceptable to the patient.Supplementary active compounds may also be incorporated into thecompositions. The “pharmaceutically acceptable carrier” may furtherinclude a pharmaceutically acceptable salt of the compound useful withinthe invention. Other additional ingredients that may be included in thepharmaceutical compositions used in the practice of the invention areknown in the art and described, for example in Remington'sPharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton,Pa.), which is incorporated herein by reference.

As used herein, the term “alkyl,” by itself or as part of anothersubstituent means, unless otherwise stated, a straight or branched chainhydrocarbon having the number of carbon atoms designated (i.e. C₁₋₆means one to six carbon atoms) and including straight, branched chain,or cyclic substituent groups. Examples include methyl, ethyl, propyl,isopropyl, butyl, isobutyl, tert-butyl, pentyl, neopentyl, hexyl, andcyclopropylmethyl. Most preferred is (C₁-C₆)alkyl, particularly ethyl,methyl, isopropyl, isobutyl, n-pentyl, n-hexyl and cyclopropylmethyl.

As used herein, the term “substituted alkyl” means alkyl as definedabove, substituted by one, two or three substituents selected from thegroup consisting of halogen, —OH, alkoxy, —NH₂, —N(CH₃)₂, —C(═O)OH,trifluoromethyl, —C≡N, —C(═O)O(C₁-C₄)alkyl, —C(═O)NH₂, —SO₂NH₂,—C(═NH)NH₂, and —NO₂, preferably containing one or two substituentsselected from halogen, —OH, alkoxy, —NH₂, trifluoromethyl, —N(CH₃)₂, and—C(═O)OH, more preferably selected from halogen, alkoxy and —OH.Examples of substituted alkyls include, but are not limited to,2,2-difluoropropyl, 2-carboxycyclopentyl and 3-chloropropyl.

As used herein, the term “heteroalkyl” by itself or in combination withanother term means, unless otherwise stated, a stable straight orbranched chain alkyl group consisting of the stated number of carbonatoms and one or two heteroatoms selected from the group consisting ofO, N, and S, and wherein the nitrogen and sulfur atoms may be optionallyoxidized and the nitrogen heteroatom may be optionally quaternized. Theheteroatom(s) may be placed at any position of the heteroalkyl group,including between the rest of the heteroalkyl group and the fragment towhich it is attached, as well as attached to the most distal carbon atomin the heteroalkyl group. Examples include: —O—CH₂—CH₂—CH₃,—CH₂—CH₂—CH₂—OH, —CH₂—CH₂—NH—CH₃, —CH₂—S—CH₂—CH₃, and —CH₂CH₂—S(═O)—CH₃.Up to two heteroatoms may be consecutive, such as, for example,—CH₂—NH—OCH₃, or —CH₂—CH₂—S—S—CH₃

As used herein, the term “alkoxy” employed alone or in combination withother terms means, unless otherwise stated, an alkyl group having thedesignated number of carbon atoms, as defined above, connected to therest of the molecule via an oxygen atom, such as, for example, methoxy,ethoxy, 1-propoxy, 2-propoxy (isopropoxy) and the higher homologs andisomers. Preferred are (C₁-C₃) alkoxy, particularly ethoxy and methoxy.

As used herein, the term “halo” or “halogen” alone or as part of anothersubstituent means, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom, preferably, fluorine, chlorine, or bromine,more preferably, fluorine or chlorine.

As used herein, the term “cycloalkyl” refers to a mono cyclic orpolycyclic non-aromatic radical, wherein each of the atoms forming thering (i.e. skeletal atoms) is a carbon atom. In one embodiment, thecycloalkyl group is saturated or partially unsaturated. In anotherembodiment, the cycloalkyl group is fused with an aromatic ring.Cycloalkyl groups include groups having from 3 to 10 ring atoms.Illustrative examples of cycloalkyl groups include, but are not limitedto, the following moieties:

Monocyclic cycloalkyls include, but are not limited to, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.Dicyclic cycloalkyls include, but are not limited to,tetrahydronaphthyl, indanyl, and tetrahydropentalene. Polycycliccycloalkyls include adamantine and norbornane. The term cycloalkylincludes “unsaturated nonaromatic carbocyclyl” or “nonaromaticunsaturated carbocyclyl” groups, both of which refer to a nonaromaticcarbocycle as defined herein, which contains at least one carbon carbondouble bond or one carbon carbon triple bond.

As used herein, the term “heterocycloalkyl” or “heterocyclyl” refers toa heteroalicyclic group containing one to four ring heteroatoms eachselected from O, Sand N. In one embodiment, each heterocycloalkyl grouphas from 4 to 10 atoms in its ring system, with the proviso that thering of said group does not contain two adjacent O or S atoms. Inanother embodiment, the heterocycloalkyl group is fused with an aromaticring. In one embodiment, the nitrogen and sulfur heteroatoms may beoptionally oxidized, and the nitrogen atom may be optionallyquaternized. The heterocyclic system may be attached, unless otherwisestated, at any heteroatom or carbon atom that affords a stablestructure. A heterocycle may be aromatic or non-aromatic in nature. Inone embodiment, the heterocycle is a heteroaryl.

An example of a 3-membered heterocycloalkyl group includes, and is notlimited to, aziridine. Examples of 4-membered heterocycloalkyl groupsinclude, and are not limited to, azetidine and a beta lactam. Examplesof 5-membered heterocycloalkyl groups include, and are not limited to,pyrrolidine, oxazolidine and thiazolidinedione. Examples of 6-memberedheterocycloalkyl groups include, and are not limited to, piperidine,morpholine and piperazine. Other non-limiting examples ofheterocycloalkyl groups are:

Examples of non-aromatic heterocycles include monocyclic groups such asaziridine oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine,pyrroline, pyrazolidine, imidazoline, dioxolane, sulfolane,2,3-dihydrofuran, 2,5-dihydrofuran, tetrahydrofuran, thiophane,piperidine, 1,2,3,6-tetrahydropyridine, 1,4-dihydropyridine, piperazine,morpholine, thiomorpholine, pyran, 2,3-dihydropyran, tetrahydropyran,1,4-dioxane, 1,3-dioxane, homopiperazine, homopiperidine, 1,3-dioxepane,4,7-dihydro-1,3-dioxepin, and hexamethyleneoxide.

As used herein, the term “aromatic” refers to a carbocycle orheterocycle with one or more polyunsaturated rings and having aromaticcharacter, i.e. having (4n+2) delocalized nt (pi) electrons, where n isan integer.

As used herein, the term “aryl,” employed alone or in combination withother terms, means, unless otherwise stated, a carbocyclic aromaticsystem containing one or more rings (typically one, two or three rings),wherein such rings may be attached together in a pendent manner, such asa biphenyl, or may be fused, such as naphthalene. Examples of arylgroups include phenyl, anthracyl, and naphthyl. Preferred examples arephenyl and naphthyl, most preferred is phenyl.

As used herein, the term “aryl-(C₁-C₃)alkyl” means a functional groupwherein a one- to three-carbon alkylene chain is attached to an arylgroup, e.g., —CH₂CH₂-phenyl. Preferred is aryl-CH₂— and aryl-CH(CH₃)—.The term “substituted aryl-(C₁-C₃)alkyl” means an aryl-(C₁-C₃)alkylfunctional group in which the aryl group is substituted. Preferred issubstituted aryl(CH₂)—. Similarly, the term “heteroaryl-(C₁-C₃)alkyl”means a functional group wherein a one to three carbon alkylene chain isattached to a heteroaryl group, e.g., —CH₂CH₂-pyridyl. Preferred isheteroaryl-(CH₂)—. The term “substituted heteroaryl-(C₁-C₃)alkyl” meansa heteroaryl-(C₁-C₃)alkyl functional group in which the heteroaryl groupis substituted. Preferred is substituted heteroaryl-(CH₂)—.

As used herein, the term “heteroaryl” or “heteroaromatic” refers to aheterocycle having aromatic character. A polycyclic heteroaryl mayinclude one or more rings that are partially saturated. Examples includethe following moieties:

Examples of heteroaryl groups also include pyridyl, pyrazinyl,pyrimidinyl (particularly 2- and 4-pyrimidinyl), pyridazinyl, thienyl,furyl, pyrrolyl (particularly 2-pyrrolyl), imidazolyl, thiazolyl,oxazolyl, pyrazolyl (particularly 3- and 5-pyrazolyl), isothiazolyl,1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl,1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl and1,3,4-oxadiazolyl.

Examples of polycyclic heterocycles and heteroaryls include indolyl(particularly 3-, 4-, 5-, 6- and 7-indolyl), indolinyl, quinolyl,tetrahydroquinolyl, isoquinolyl (particularly 1- and 5-isoquinolyl),1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl (particularly 2-and 5-quinoxalinyl), quinazolinyl, phthalazinyl, 1,8-naphthyridinyl,1,4-benzodioxanyl, coumarin, dihydrocoumarin, 1,5-naphthyridinyl,benzofuryl (particularly 3-, 4-, 5-, 6- and 7-benzofuryl),2,3-dihydrobenzofuryl, 1,2-benzisoxazolyl, benzothienyl (particularly3-, 4-, 5-, 6-, and 7-benzothienyl), benzoxazolyl, benzothiazolyl(particularly 2-benzothiazolyl and 5-benzothiazolyl), purinyl,benzimidazolyl (particularly 2-benzimidazolyl), benzotriazolyl,thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrrolizidinyl, andquinolizidinyl.

As used herein, the term “substituted” means that an atom or group ofatoms has replaced hydrogen as the substituent attached to anothergroup. The term “substituted” further refers to any level ofsubstitution, namely mono-, di-, tri-, tetra-, or penta-substitution,where such substitution is permitted. The substituents are independentlyselected, and substitution may be at any chemically accessible position.In one embodiment, the substituents vary in number between one and four.In another embodiment, the substituents vary in number between one andthree. In yet another embodiment, the substituents vary in numberbetween one and two.

As used herein, the term “optionally substituted” means that thereferenced group may be substituted or unsubstituted. In one embodiment,the referenced group is optionally substituted with zero substituents,i.e., the referenced group is unsubstituted. In another embodiment, thereferenced group is optionally substituted with one or more additionalgroup(s) individually and independently selected from groups describedherein.

In one embodiment, the substituents are independently selected from thegroup consisting of oxo, halogen, —CN, —NH₂, —OH, —NH(CH₃), —N(CH₃)₂,alkyl (including straight chain, branched and/or unsaturated alkyl),substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, fluoro alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted alkoxy, fluoroalkoxy,—S-alkyl, S(═O)₂alkyl, —C(═O)NH[substituted or unsubstituted alkyl, orsubstituted or unsubstituted phenyl], —C(═O)N[H or alkyl]₂,—OC(═O)N[substituted or unsubstituted alkyl]₂, —NHC(═O)NH[substituted orunsubstituted alkyl, or substituted or unsubstituted phenyl],—NHC(═O)alkyl, —N[substituted or unsubstituted alkyl]C(═O)[substitutedor unsubstituted alkyl], —NHC(═O)[substituted or unsubstituted alkyl],—C(OH)[substituted or unsubstituted alkyl]₂, and —C(NH₂)[substituted orunsubstituted alkyl]₂. In another embodiment, by way of example, anoptional substituent is selected from oxo, fluorine, chlorine, bromine,iodine, —CN, —NH₂, —OH, —NH(CH₃), —N(CH₃)₂, —CH₃, —CH₂CH₃, —CH(CH₃)₂,—CF₃, —CH₂CF₃, —OCH₃, —OCH₂CH₃, —OCH(CH₃)₂, —OCF₃, —OCH₂CF₃,—S(═O)₂—CH₃, —C(═O)NH₂, —C(═O)—NHCH₃, —NHC(═O)NHCH₃, —C(═O)CH₃, and—C(═O)OH. In yet one embodiment, the substituents are independentlyselected from the group consisting of C₁₋₆ alkyl, —OH, C₁₋₆ alkoxy,halo, amino, acetamido, oxo and nitro. In yet another embodiment, thesubstituents are independently selected from the group consisting ofC₁₋₆ alkyl, C₁₋₆ alkoxy, halo, acetamido, and nitro. As used herein,where a substituent is an alkyl or alkoxy group, the carbon chain may bebranched, straight or cyclic, with straight being preferred.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible sub-ranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

DESCRIPTION

The present invention relates to the unexpected discovery of novelcompounds that bind to and modulate the activity of the Sigma receptor.In one embodiment, the Sigma receptor modulators of the invention areSigma receptor antagonists. In another embodiment, the Sigma receptor isselected from the group consisting of Sigma1, Sigma2 and a combinationthereof. In yet another embodiment, the Sigma receptor is Sigma1.

The compounds of the invention may be used to treat Sigmareceptor-related diseases and disorders, such as but not limited tocancer, neuropathic pain, depression, substance abuse, epilepsy,psychosis, Alzheimer's disease, Parkinson's disease, frontotemporallobar degeneration (FTLD), amyotrophic lateral sclerosis (ALS) andcombinations thereof. The cancer may be selected from the groupconsisting of prostate cancer, liver cancer, pancreas cancer, breastcancer, neuroblastoma, leukemia, CNS cancers (including brain tumors),and combinations thereof. In one embodiment, the therapeutic effectelicited by the compounds of the invention is mediated by the Sigmareceptor. In another embodiment, the therapeutic effect elicited by thecompounds of the invention is not mediated by the Sigma receptor.

Without wishing to be limited by theory, proteinhomeostasis/“proteostasis” (i.e., maintenance of proper proteinsynthesis, processing, folding. transport, assembly, and degradation)modulating properties of the compounds of the invention allow then to beused in the treatment of any disease in which protein homeostasis isdisrupted (e.g., neurodegenerative diseases) or in which this process isespecially crucial (e.g., cancer). In one embodiment, the compound ofthe invention crosses the blood-brain barrier. In another embodiment,the compound of the invention does not cross the blood-brain barrier.

In a non-limiting aspect, the present invention relates to theunexpected discovery that Sigma1 antagonists induces ER stress (such astranslation arrest, unfolded protein response (UPR), or autophagy) andactivates the unfolded protein response (UPR) in a dose and timeresponsive manner. As demonstrated herein, autophagy was engagedfollowing extended treatment with Sigma1 antagonists, suggesting thatprotracted UPR results in autophagy as a secondary response. In fact,UPR activation preceded autophagosome formation and autophagy precededapoptosis in Sigma1 antagonist-treated cells. Inhibition of Sigma1antagonist-induced UPR or autophagy accelerated Sigma1antagonist-mediated apoptosis. Therefore, as demonstrate herein, thecombination of a Sigma1 antagonist with an agent targeting the UPRand/or autophagic survival pathways provides a novel and efficaciousapproach to treat, ameliorate or prevent cancer. In one embodiment, thecompounds of the present invention induce endoplasmic reticulum (ER)stress, such as, but are not limited to, translation arrest, unfoldedprotein response (UPR), autophagy, and combinations thereof. In anotherembodiment, the compounds of the present invention modulate cellularprotein ubiquitylation, including but not being limited to ER associatedproteasomal degradation (ERAD). As demonstrate herein, the Sigma ligand,IPAG, induced a novel, ubiquitin-selective autophagy in breast cancercell lines.

The present invention includes a composition comprising at least onecompound of the invention, wherein the composition optionally furthercomprise at least one additional therapeutic agent. The presentinvention also includes a composition comprising a Sigmareceptor-modulating compound and at least one additional therapeuticagent. In one embodiment, the additional therapeutic agent targets theUPR and/or autophagic survival pathway. In another embodiment, theadditional therapeutic agent binds to and modulates the Sigma receptor.In yet another embodiment, the additional therapeutic agent is achemotherapeutic and/or hormone therapy agent.

Examples of additional therapeutic agents contemplated within theinvention include, but are not limited to, growth factor receptorinhibitors, monoclonal antibodies against growth factor receptors (e.g.,Traztuzumab), hormone receptor antagonists (e.g., androgen receptorinhibitors), autophagy modulators (such as rapamycin and its analogs or“rapalogs”), ER stress response inhibitors, proteasome inhibitors,p97/VCP inhibitors (e.g., DBeQ and derivatives thereof—Chou et al.,2011, Proc. Natl. Acad. Sci. USA 108(12):4834-9), and combinationsthereof. Non-limiting examples of additional therapeutic agentscontemplated within the invention include octapeptide, somatostatin,analoguem, lanreotide, angiopeptin, dermopeptin, octreotide,pegvisomant, 3-methyladenine, chloroquine, hydroxychloroquine,wortmannin, eeyarestatin I, salubrinal, versipelostatin,2H-isoindole-2-carboxylic acid, 4-fluoro-1,3-dihydro-(2R,6S, 12Z,13aS,14aR,16aS)-14a-[[(cyclopropylsulfonyl)amino]carbonyl]-6-[[(1,1-dimethylethoxy)carbonyl]amino]-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a-hexadecahydro-5,16-dioxocyclopropa[e]pyrrolo[1,2-a][1,4]diazacyclopentadecin-2-ylester (Danoprevir),adamantane-acetyl-(6-aminohexanoyl)3-(leucinyl)3-vinyl-(methyl)-sulfone,N-acetyl-L-leucyl-L-leucyl-L-methional,N-[(phenylmethoxy)carbonyl]-L-leucyl-N-[(1S)-1-formyl-3-methylbutyl]-L-leucinamide, (2R,3S,4R)-3-hydroxy-2-[(1S)-1-hydroxy-2-methylpropyl]-4-methyl-5-oxo-2-pyrrolidinecarboxy-N-acetyl-L-cysteinethioester, N—[N—(N-acetyl-L-leucyl)-L-leucyl]-L-norleucine, lactacystin,4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride,(S)-1-carboxy-2-phenyl]-carbamoyl-Arg-Val-arginal, bovine pancreatictrypsin inhibitor, [(2S,2R)-3-amino-2-hydroxy-4-phenylbutanoyl]-L-leucine,N—[(S)-1-carboxy-isopentyl)-carbamoyl-alpha-(2-iminohexahydro-4-(S)-pyrimidyl]-L-glycyl-L-phenylalaninal,ethylenediamine-tetraacetic acid disodium salt dehydrate,acetyl-leucyl-leucyl-arginal, isovaleryl-Val-Val-AHMHA-Ala-AHMHA whereAHMHA=(3 S,4S)-4-amino-3-hydroxy-6-methylheptanoic acid,N-alpha-L-rhamnopyranosyloxy-(hydroxyphosphinyl)-L-leucyl-L-tryptophan,phenylmethanesulfonyl fluoride, bortezomib, carfilzomib, ONX 0912,NPI-0052, CEP-18770, MLN9708, disulfiram, epigallocatechin-3-gallate,salinosporamide A, PI3K inhibitors, lapatinib, rapamycin and rapalogs,heat shock protein (HSP) inhibitors (e.g., geldanamycin and derivativessuch as 17-AAG), androgen receptor inhibitors (e.g., MDV3100, ARN-509),and conjugation products of Sigma ligands with targeting components suchas Herceptin/Traztuzumab (e.g., Trastuzumab-emtansine, T-DM1, is anantibody-drug conjugate comprising the antibody trastuzumab (Herceptin)linked to the cytotoxin mertansine—Niculescu-Duvaz, 2010, Curr. Opin.Mol. Ther. 12(3):350-60).

The compounds of the present invention, used alone or in combinationwith at least one additional therapeutic agent (e.g., those that targetthe ubiquitin proteasome system (UPS) and/or autophagic survivalpathways), are useful in the treatment of Sigma receptor-relateddisorders or diseases. Examples of disorders or diseases contemplatedwithin the invention include, but are not limited to, cancer,neuropathic pain, depression, substance abuse, epilepsy, psychosis,Alzheimer's disease, Parkinson's disease, neurodegeneration, lysosomalstorage disease, diseases in which protein folding and processing isaltered, and indications wherein the modulation of autophagy may betherapeutically beneficial. In a preferred embodiment, the disease iscancer.

In one embodiment, the compounds of the present invention have improveddrug-like properties over compounds known in the art to bind to andmodulate the Sigma receptor. In another embodiment, the compounds of thepresent invention do not cross the blood-brain barrier. In yet anotherembodiment, the compounds of the present invention cross the blood-brainbarrier.

The compounds of the present invention include a Sigma ligand probe,which may be used to study biological phenomena in living cells. In oneembodiment, the Sigma ligand probe comprises a fluorophore. In anotherembodiment, the fluorophore is 7-amino-4-methyl coumarin (FIGS.64A-64C). In one embodiment, the fluorescent probe is2-(4-(4-methyl-2-oxo-2H-chromen-7-yl)piperazin-1-yl)ethyl1-phenylcyclohexanecarboxylate. In another embodiment, the fluorescentprobe is1-(4-methoxyphenyl)-3-(3-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)propyl)guanidine.In yet another embodiment, the fluorescent probe is1-(4-iodophenyl)-3-(3-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)propyl)guanidine.In yet another embodiment, the fluorescent probe is1-(3-(4-fluorophenoxy)propyl)-3-(4-methyl-2-oxo-2H-chromen-7-yl)guanidine).

The compounds of the present invention may be characterized bypharmacological, cellular, biochemical, in vivo, pharmacokinetics, orpharmacodynamics properties. Preferred examples of characterizationstudies include, but are not limited to, Sigma1-ligand bindingproperties, signaling pathway analysis and/or characterization,proteomic analysis of Sigma1 protein associations in response to Sigmaligand treatment, tumor, brain response, and toxicity.

Compounds of the Invention

The compounds of the present invention may be synthesized usingtechniques well-known in the art of organic synthesis. The startingmaterials and intermediates required for the synthesis may be obtainedfrom commercial sources or synthesized according to methods known tothose skilled in the art.

In one aspect, the compound of the invention is a compound of formula(I), or a salt, solvate, or N-oxide thereof:

wherein:

ring A is a monocyclic or bicyclic aryl or a monocyclic or bicyclicheteroaryl ring, and wherein the aryl or heteroaryl ring is optionallysubstituted with 0-4 R¹ groups;

each occurrence of R¹ is independently selected from the groupconsisting of —C₁-C₆ alkyl, —C₁-C₆ fluoroalkyl, —C₁-C₆ heteroalkyl, F,Cl, Br, I, —CN, —NO₂, —OR³, —SR³, —S(═O)R³, —S(═O)₂R³, —NHS(═O)₂R³,—C(═O)R³, —OC(═O)R³, —CO₂R³, —OCO₂R³, —CH(R³)₂, —N(R³)₂, —C(═O)N(R³)₂,—OC(═O)N(R³)₂, —NHC(═O)NH(R³), —NHC(═O)R³, —NHC(═O)OR³, —C(OH)(R³)₂, and—C(NH₂)(R³)₂;

each occurrence of R² is independently selected from the groupconsisting of H, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, and —C₁-C₃ alkyl-(C₃-C₆cycloalkyl), wherein the alkyl, heteroalkyl or cycloalkyl group isoptionally substituted with 0-5 R¹ groups, or X³ and R² combine to forma (C₃-C₇)heterocycloalkyl group, optionally substituted with 0-2 R¹groups;

each occurrence of R³ is independently selected from the groupconsisting of H, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, aryl, and —C₁-C₃alkyl-(C₃-C₆ cycloalkyl), wherein the alkyl, heteroalkyl, aryl, orcycloalkyl group is optionally substituted with 0-5 R¹ groups;

X¹ is —CH₂—, —S—, —O— or —(NR²)—;

X² is ═CH₂, ═S, ═O or ═NR²; and

X³ is —S—, —O—, or —NR²⁻.

In one embodiment, ring A is a monocyclic aryl or monocyclic heteroarylring optionally substituted with 0-4 R¹ groups. In another embodiment,ring A is unsubstituted. In yet another embodiment, ring A is phenyl orsubstituted phenyl.

In a preferred embodiment, X¹ and X³ are both —NH—, and X² is ═NH.

In another aspect, the compound of the invention is a compound offormula (II), or a salt, solvate, or N-oxide thereof:

R^(A)—R^(B)  (II), wherein;

R^(A) is selected from the group consisting of

wherein

X⁴ is selected from the group consisting of F, Cl, Br, and I; and

R^(B) is selected from the group consisting of:

In another aspect, the compound of the invention is a compound offormula (III), or a salt, solvate, or N-oxide thereof:

wherein within formula (III);

each occurrence of R¹ and R² is independently selected from the groupconsisting of —C₁-C₆ alkyl, —C₁-C₆ fluoroalkyl, —C₁-C₆ heteroalkyl, F,Cl, Br, I, —CN, —NO₂, —OR⁵, —SR⁵, —S(═O)R⁵, —S(═O)₂R⁵, —NHS(═O)₂R⁵,—C(═O)R⁵, —OC(═O)R⁵, —CO₂R⁵, —OCO₂R⁵, —CH(R)₂, —N(R⁵)₂, —C(═O)N(R)₂,—OC(═O)N(R)₂, —NHC(═O)NH(R⁵), —NHC(═O)R⁵, —NHC(═O)OR⁵, —C(OH)(R⁵)₂, and—C(NH₂)(R⁵)₂;

R³ is selected from the group consisting of —C₁-C₆ alkyl, —C₁-C₆fluoroalkyl, —C₁-C₆ alkoxy, F, Cl, Br, and I;

R⁴ is selected from the group consisting of —C₁-C₆ alkyl, —C₁-C₆ alkoxy,F, Cl, Br, and I;

each occurrence of R⁵ is independently selected from the groupconsisting of H, C₁-C₆ alkyl, C₁-C₆ heteroalkyl, aryl, and —C₁-C₃alkyl-(C₃-C₆ cycloalkyl), wherein the alkyl, heteroalkyl, aryl, orcycloalkyl group is optionally substituted.

X is selected from the group consisting of CH₂, C═O, or O;

n is an integer from 1-3;

x is an integer from 0-4; and

y is an integer from 0-4.

In one embodiment, the compound of the invention is selected from thegroup consisting of:

-   1-(3-(4-fluorophenoxy)propyl)-3-(4-iodophenyl)guanidine (Compound A;    also known as JMS-51-58 or 51-58);-   1-(3-(4-fluorophenoxy)propyl)-3-(4-methoxyphenyl)guanidine (Compound    B);-   1-(n-propyl)-3-(4-iodophenyl)guanidine (Compound C);-   1-(n-propyl)-3-(4-methoxyphenyl)guanidine (Compound D);-   1,3-bis(3-(4-fluorophenoxy)propyl)guanidine (Compound E);-   1-(3-(4-fluorophenoxy)propyl)-3-(4-trifluoromethylphenyl)guanidine    (Compound F);-   1-(3-(4-fluorophenoxy)propyl)-3-(4-chlorophenyl)guanidine (Compound    G);-   1-(3-(4-fluorophenoxy)propyl)-3-(4-methyl-2-oxo-2H-chromen-7-yl)guanidine    (Compound H);    a salt, solvate or N-oxide thereof; and any combinations thereof.

Preparation of the Compounds of the Invention

Compounds of Formula (I) may be prepared by the general schemesdescribed herein, using the synthetic method known by those skilled inthe art. The following examples illustrate non-limiting embodiments ofthe invention.

In a non-limiting embodiment, the synthesis of unsymmetricalN,N′-disubstituted guanidines is accomplished by coupling an arylcyanamide and an amine (FIG. 17). In one embodiment, the couplingreaction takes place at an elevated temperature ranging from 80° C. to250° C. An aniline may be converted to an aryl cyanamide with cyanogenbromide in ether. The unsymmetrical N,N′-disubstituted guanidine is thenformed by coupling the aryl cyanamide with an amine. Non-limitingexamples of coupling methods include heating in acetonitrile at reflux,and heating at 120° C. in a microwave.

In another non-limiting embodiment, unsymmetrical N,N′-disubstitutedguanidines may be synthesized by coupling a benzimidothioate and anamine (FIG. 18). For example, an aniline may be reacted with potassiumisothiocyanate to provide a thiourea. The thiourea may then be treatedwith methyl iodide in acetone heated to reflux, providing the desiredbenzimidothioate. The unsymmetrical N,N′-disubstituted guanidine maythen be formed by coupling the benzimidothioate with an amine. Anon-limiting example of a coupling method includes heating in ethanol atreflux.

The compounds of the invention may possess one or more stereocenters,and each stereocenter may exist independently in either the R or Sconfiguration. In one embodiment, compounds described herein are presentin optically active or racemic forms. It is to be understood that thecompounds described herein encompass racemic, optically-active,regioisomeric and stereoisomeric forms, or combinations thereof thatpossess the therapeutically useful properties described herein.Preparation of optically active forms is achieved in any suitablemanner, including by way of non-limiting example, by resolution of theracemic form with recrystallization techniques, synthesis fromoptically-active starting materials, chiral synthesis, orchromatographic separation using a chiral stationary phase. In oneembodiment, a mixture of one or more isomer is utilized as thetherapeutic compound described herein. In another embodiment, compoundsdescribed herein contain one or more chiral centers. These compounds areprepared by any means, including stereoselective synthesis,enantioselective synthesis and/or separation of a mixture of enantiomersand/or diastereomers. Resolution of compounds and isomers thereof isachieved by any means including, by way of non-limiting example,chemical processes, enzymatic processes, fractional crystallization,distillation, and chromatography.

The methods and formulations described herein include the use ofN-oxides (if appropriate), crystalline forms (also known as polymorphs),solvates, amorphous phases, and/or pharmaceutically acceptable salts ofcompounds having the structure of any compound of the invention, as wellas metabolites and active metabolites of these compounds having the sametype of activity. Solvates include water, ether (e.g., tetrahydrofuran,methyl tert-butyl ether) or alcohol (e.g., ethanol) solvates, acetatesand the like. In one embodiment, the compounds described herein exist insolvated forms with pharmaceutically acceptable solvents such as water,and ethanol. In another embodiment, the compounds described herein existin unsolvated form.

In one embodiment, the compounds of the invention may exist astautomers. All tautomers are included within the scope of the compoundspresented herein.

In one embodiment, compounds described herein are prepared as prodrugs.A “prodrug” refers to an agent that is converted into the parent drug invivo. In one embodiment, upon in vivo administration, a prodrug ischemically converted to the biologically, pharmaceutically ortherapeutically active form of the compound. In another embodiment, aprodrug is enzymatically metabolized by one or more steps or processesto the biologically, pharmaceutically or therapeutically active form ofthe compound.

In one embodiment, sites on, for example, the aromatic ring portion ofcompounds of the invention are susceptible to various metabolicreactions. Incorporation of appropriate substituents on the aromaticring structures may reduce, minimize or eliminate this metabolicpathway. In one embodiment, the appropriate substituent to decrease oreliminate the susceptibility of the aromatic ring to metabolic reactionsis, by way of example only, a deuterium, a halogen, or an alkyl group.

Compounds described herein also include isotopically-labeled compoundswherein one or more atoms is replaced by an atom having the same atomicnumber, but an atomic mass or mass number different from the atomic massor mass number usually found in nature. Examples of isotopes suitablefor inclusion in the compounds described herein include and are notlimited to ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ³⁶Cl, ¹⁸F, ¹²³I, ¹²⁵I, ^(13N), ¹⁵N,¹⁵O, ¹⁷O, ¹⁸O, ³²P, and ³⁵S. In one embodiment, isotopically-labeledcompounds are useful in drug and/or substrate tissue distributionstudies. In another embodiment, substitution with heavier isotopes suchas deuterium affords greater metabolic stability (for example, increasedin vivo half-life or reduced dosage requirements). In yet anotherembodiment, substitution with positron emitting isotopes, such as ¹¹C,¹⁸F, ¹⁵O and ¹³N, is useful in Positron Emission Topography (PET)studies for examining substrate receptor occupancy. Isotopically-labeledcompounds are prepared by any suitable method or by processes using anappropriate isotopically-labeled reagent in place of the non-labeledreagent otherwise employed.

In one embodiment, the compounds described herein are labeled by othermeans, including, but not limited to, the use of chromophores orfluorescent moieties, bioluminescent labels, or chemiluminescent labels.

The compounds described herein, and other related compounds havingdifferent substituents are synthesized using techniques and materialsdescribed herein and as described, for example, in Fieser & Fieser'sReagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons,1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 andSupplementals (Elsevier Science Publishers, 1989); Organic Reactions,Volumes 1-40 (John Wiley and Sons, 1991), Larock's Comprehensive OrganicTransformations (VCH Publishers Inc., 1989), March, Advanced OrganicChemistry 4^(th) Ed., (Wiley 1992); Carey & Sundberg, Advanced OrganicChemistry 4th Ed., Vols. A and B (Plenum 2000,2001), and Green & Wuts,Protective Groups in Organic Synthesis 3rd Ed., (Wiley 1999) (all ofwhich are incorporated by reference for such disclosure). Generalmethods for the preparation of compound as described herein are modifiedby the use of appropriate reagents and conditions, for the introductionof the various moieties found in the formula as provided herein.

Compounds described herein are synthesized using any suitable proceduresstarting from compounds that are available from commercial sources, orare prepared using procedures described herein.

In one embodiment, reactive functional groups, such as hydroxyl, amino,imino, thio or carboxy groups, are protected in order to avoid theirunwanted participation in reactions. Protecting groups are used to blocksome or all of the reactive moieties and prevent such groups fromparticipating in chemical reactions until the protective group isremoved. In another embodiment, each protective group is removable by adifferent means. Protective groups that are cleaved under totallydisparate reaction conditions fulfill the requirement of differentialremoval.

In one embodiment, protective groups are removed by acid, base, reducingconditions (such as, for example, hydrogenolysis), and/or oxidativeconditions. Groups such as trityl, dimethoxytrityl, acetal andt-butyldimethylsilyl are acid labile and are used to protect carboxy andhydroxy reactive moieties in the presence of amino groups protected withCbz groups, which are removable by hydrogenolysis, and Fmoc groups,which are base labile. Carboxylic acid and hydroxy reactive moieties areblocked with base labile groups such as, but not limited to, methyl,ethyl, and acetyl, in the presence of amines that are blocked with acidlabile groups, such as t-butyl carbamate, or with carbamates that areboth acid and base stable but hydrolytically removable.

In one embodiment, carboxylic acid and hydroxy reactive moieties areblocked with hydrolytically removable protective groups such as thebenzyl group, while amine groups capable of hydrogen bonding with acidsare blocked with base labile groups such as Fmoc. Carboxylic acidreactive moieties are protected by conversion to simple ester compoundsas exemplified herein, which include conversion to alkyl esters, or areblocked with oxidatively-removable protective groups such as2,4-dimethoxybenzyl, while co-existing amino groups are blocked withfluoride labile silyl carbamates.

Allyl blocking groups are useful in the presence of acid- andbase-protecting groups since the former are stable and are subsequentlyremoved by metal or pi-acid catalysts. For example, an allyl-blockedcarboxylic acid is deprotected with a palladium-catalyzed reaction inthe presence of acid labile t-butyl carbamate or base-labile acetateamine protecting groups. Yet another form of protecting group is a resinto which a compound or intermediate is attached. As long as the residueis attached to the resin, that functional group is blocked and does notreact. Once released from the resin, the functional group is availableto react.

Typically blocking/protecting groups may be selected from:

Other protecting groups, plus a detailed description of techniquesapplicable to the creation of protecting groups and their removal aredescribed in Greene & Wuts, Protective Groups in Organic Synthesis, 3rdEd., John Wiley & Sons, New York, N.Y., 1999, and Kocienski, ProtectiveGroups, Thieme Verlag, New York, N.Y., 1994, which are incorporatedherein by reference for such disclosure.

Methods of the Invention

The invention includes a method of treating, ameliorating or preventinga Sigma compound of the invention. In one embodiment, the Sigmareceptor-related disease or disorder is selected from the groupcomprising cancer, neuropathic pain, depression, substance abuse,epilepsy, psychosis, Alzheimer's disease, Parkinson's disease, andcombinations thereof. In carernce r c cer, breast cancer, CNS tumors(including brain tumors), neuroblastoma, leukemia, and inations thereof.

The invention also includes a method of treating, ameliorating orpreventing am Sigma receptor-related disorder or disease in a subject inneed thereof. The method comprises administering to the subject aneffective amount of a therapeutic composition comprising a Sigmareceptor-modulating compound, and further administering to the subject atherapeutic agent that inhibits the ubiquitin proteasome system (UPS)and/or autophagic survival pathways. In one embodiment, the Sigmareceptor-modulating compound is a compound of the invention.

In one embodiment, administering the Sigma receptor-modulating compoundto the subject allows for administering a lower dose of the therapeuticagent that inhibits the ubiquitin proteasome system (UPS) and/orautophagic survival pathways, as compared to the dose of the therapeuticagent alone that is required to achieve similar results in treating,ameliorating or preventing the Sigma receptor-related disorder in thesubject. In another embodiment, the Sigma receptor-modulating compoundand the therapeutic agent are co-administered to the subject. In yetanother embodiment, the Sigma receptor-modulating compound and thetherapeutic agent are co-formulated and co-administered to the subject.

In one embodiment, the methods described herein further compriseinhibiting the Sigma receptor. In another embodiment, the methodsdescribed herein further comprise modulating the Sigma receptor.

In one embodiment, the subject is a mammal. In another embodiment, themammal is a human.

Combination Therapies

The compounds of the present invention are intended to be useful incombination with one or more additional compounds. These additionalcompounds may comprise compounds of the present invention or therapeuticagents known to treat, prevent, or reduce the symptoms or effects ofSigma receptor-related disorders or diseases. Such compounds include,but are not limited to, hormone receptor antagonists, autophagyinhibitors, ER stress response inhibitors, and proteasome inhibitors.

In non-limiting examples, the compounds of the invention may be used incombination with one or more therapeutic agents (or a salt, solvate orprodrug thereof) selected from the group consisting of

hormone receptor antagonists, including but are not limited tooctapeptide, somatostatin, analoguem, lanreotide, angiopeptin,dermopeptin, octreotide, and pegvisomant;

autophagy inhibitors, including but are not limited to 3-methyladenine,chloroquine, hydroxychloroquine, and wortmannin;

ER stress response inhibitors, including but are not limited toeeyarestatin I, salubrinal, and versipelostatin;

proteasome inhibitors, including but are not limited to2H-isoindole-2-carboxylic acid, 4-fluoro-1,3-dihydro-(2R,6S, 12Z,13aS,14aR,16aS)-14a-[[(cyclopropylsulfonyl)amino]carbonyl]-6-[[(1,1-dimethylethoxy)carbonyl]amino]-1,2,3,5,6,7,8,9,10,11,13a,14,14a,15,16,16a-hexadecahydro-5,16-dioxocyclopropa[e]pyrrolo[1,2-a][1,4]diazacyclopentadecin-2-ylester (Danoprevir),adamantane-acetyl-(6-aminohexanoyl)3-(leucinyl)3-vinyl-(methyl)-sulfone,N-acetyl-L-leucyl-L-leucyl-L-methional,N-[(phenylmethoxy)carbonyl]-L-leucyl-N-[(1S)-1-formyl-3-methylbutyl]-L-leucinamide,(2R,3S,4R)-3-hydroxy-2-[(1S)-1-hydroxy-2-methylpropyl]-4-methyl-5-oxo-2-pyrrolidinecarboxy-N-acetyl-L-cysteinethioester, N—[N—(N-acetyl-L-leucyl)-L-leucyl]-L-norleucine, lactacystin,4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride,(S)-1-carboxy-2-phenyl]-carbamoyl-arg-val-arginal, bovine pancreatictrypsin inhibitor, [(2S,2R)-3-amino-2-hydroxy-4-phenylbutanoyl]-L-leucine,N—[(S)-1-carboxy-isopentyl)-carbamoyl-alpha-(2-iminohexahydro-4-(S)-pyrimidyl]-L-glycyl-L-phenylalaninal,ethylenediamine-tetraacetic acid disodium salt dehydrate,acetyl-leucyl-leucyl-arginal, isovaleryl-val-val-AHMHA-ala-AHMHA whereAHMHA=(3 S, 4S)-4-amino-3-hydroxy-6-methylheptanoic acid,N-alpha-L-rhamnopyranosyloxy(hydroxyphosphinyl)-L-leucyl-L-tryptophan,phenylmethanesulfonyl fluoride, bortezomib, carfilzomib, ONX 0912,NPI-0052, CEP-18770, MLN9708, disulfiram, epigallocatechin-3-gallate,and salinosporamide A; and

p97/VCP inhibitors, including but not limited to DBeQ and derivativesthereof.

A synergistic effect may be calculated, for example, using suitablemethods such as, for example, the Sigmoid-E_(max) equation (Holford &Scheiner, 1981, Clin. Pharmacokinet. 6:429-453), the equation of Loeweadditivity (Loewe & Muischnek, 1926, Arch. Exp. Pathol Pharmacol.114:313-326) and the median-effect equation (Chou & Talalay, 1984, Adv.Enzyme Regul. 22:27-55). Each equation referred to above may be appliedto experimental data to generate a corresponding graph to aid inassessing the effects of the drug combination. The corresponding graphsassociated with the equations referred to above are theconcentration-effect curve, isobologram curve and combination indexcurve, respectively.

Administration/Dosage/Formulations

The regimen of administration may affect what constitutes an effectiveamount. The therapeutic formulations may be administered to the subjecteither prior to or after the onset of a Sigma-receptor related disorderor disease. Further, several divided dosages, as well as staggereddosages may be administered daily or sequentially, or the dose may becontinuously infused, or may be a bolus injection. Further, the dosagesof the therapeutic formulations may be proportionally increased ordecreased as indicated by the exigencies of the therapeutic orprophylactic situation.

Administration of the compositions of the present invention to apatient, preferably a mammal, more preferably a human, may be carriedout using known procedures, at dosages and for periods of time effectiveto treat Sigma-receptor related disorders or diseases in the patient. Aneffective amount of the therapeutic compound necessary to achieve atherapeutic effect may vary according to factors such as the state ofthe disease or disorder in the patient; the age, sex, and weight of thepatient; and the ability of the therapeutic compound to treatSigma-receptor related disorders or diseases in the patient. Dosageregimens may be adjusted to provide the optimum therapeutic response.For example, several divided doses may be administered daily or the dosemay be proportionally reduced as indicated by the exigencies of thetherapeutic situation. A non-limiting example of an effective dose rangefor a therapeutic compound of the invention is from about 1 and 5,000mg/kg of body weight/per day. One of ordinary skill in the art would beable to study the relevant factors and make the determination regardingthe effective amount of the therapeutic compound without undueexperimentation.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

In particular, the selected dosage level depends upon a variety offactors including the activity of the particular compound employed, thetime of administration, the rate of excretion of the compound, theduration of the treatment, other drugs, compounds or materials used incombination with the compound, the age, sex, weight, condition, generalhealth and prior medical history of the patient being treated, and likefactors well, known in the medical arts.

A medical doctor, e.g., physician or veterinarian, having ordinary skillin the art may readily determine and prescribe the effective amount ofthe pharmaceutical composition required. For example, the physician orveterinarian could start doses of the compounds of the inventionemployed in the pharmaceutical composition at levels lower than thatrequired in order to achieve the desired therapeutic effect andgradually increase the dosage until the desired effect is achieved.

In particular embodiments, it is especially advantageous to formulatethe compound in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the patients tobe treated; each unit containing a predetermined quantity of therapeuticcompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical vehicle. The dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the therapeutic compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding/formulating such a therapeutic compound for thetreatment of Sigma-receptor related disorders or diseases in a patient.

In one embodiment, the compositions of the invention are formulatedusing one or more pharmaceutically acceptable excipients or carriers. Inone embodiment, the pharmaceutical compositions of the inventioncomprise a therapeutically effective amount of a compound of theinvention and a pharmaceutically acceptable carrier.

The carrier may be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity may be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms may be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it is preferable to include isotonic agents, for example, sugars,sodium chloride, or polyalcohols such as mannitol and sorbitol, in thecomposition. Prolonged absorption of the injectable compositions may bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate or gelatin. In oneembodiment, the pharmaceutically acceptable carrier is not DMSO alone.

In one embodiment, the compositions of the invention are administered tothe patient in dosages that range from one to five times per day ormore. In another embodiment, the compositions of the invention areadministered to the patient in range of dosages that include, but arenot limited to, once every day, every two, days, every three days toonce a week, and once every two weeks. It is readily apparent to oneskilled in the art that the frequency of administration of the variouscombination compositions of the invention varies from individual toindividual depending on many factors including, but not limited to, age,disease or disorder to be treated, gender, overall health, and otherfactors. Thus, the invention should not be construed to be limited toany particular dosage regime and the precise dosage and composition tobe administered to any patient is determined by the attending physicaltaking all other factors about the patient into account.

Compounds of the invention for administration may be in the range offrom about 1 μg to about 10,000 mg, about 20 μg to about 9,500 mg, about40 μg to about 9,000 mg, about 75 μg to about 8,500 mg, about 150 μg toabout 7,500 mg, about 200 μg to about 7,000 mg, about 3050 μg to about6,000 mg, about 500 μg to about 5,000 mg, about 750 μg to about 4,000mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg toabout 1,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80mg to about 500 mg, and any and all whole or partial incrementstherebetween.

In some embodiments, the dose of a compound of the invention is fromabout 1 mg and about 2,500 mg. In some embodiments, a dose of a compoundof the invention used in compositions described herein is less thanabout 10,000 mg, or less than about 8,000 mg, or less than about 6,000mg, or less than about 5,000 mg, or less than about 3,000 mg, or lessthan about 2,000 mg, or less than about 1,000 mg, or less than about 500mg, or less than about 200 mg, or less than about 50 mg. Similarly, insome embodiments, a dose of a second compound as described herein isless than about 1,000 mg, or less than about 800 mg, or less than about600 mg, or less than about 500 mg, or less than about 400 mg, or lessthan about 300 mg, or less than about 200 mg, or less than about 100 mg,or less than about 50 mg, or less than about 40 mg, or less than about30 mg, or less than about 25 mg, or less than about 20 mg, or less thanabout 15 mg, or less than about 10 mg, or less than about 5 mg, or lessthan about 2 mg, or less than about 1 mg, or less than about 0.5 mg, andany and all whole or partial increments thereof.

In one embodiment, the present invention is directed to a packagedpharmaceutical composition comprising a container holding atherapeutically effective amount of a compound of the invention, aloneor in combination with a second pharmaceutical agent; and instructionsfor using the compound to treat, prevent, or reduce one or more symptomsof Sigma-receptor related disorders or diseases in a patient.

Formulations may be employed in admixtures with conventional excipients,i.e., pharmaceutically acceptable organic or inorganic carriersubstances suitable for oral, parenteral, nasal, intravenous,subcutaneous, enteral, or any other suitable mode of administration,known to the art. The pharmaceutical preparations may be sterilized andif desired mixed with auxiliary agents, e.g., lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure buffers, coloring, flavoring and/or aromatic substances and thelike. They may also be combined where desired with other active agents,e.g., other analgesic agents.

Routes of administration of any of the compositions of the inventioninclude oral, nasal, rectal, intravaginal, parenteral, buccal,sublingual or topical. The compounds for use in the invention may beformulated for administration by any suitable route, such as for oral orparenteral, for example, transdermal, transmucosal (e.g., sublingual,lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- andperivaginally), (intra)nasal and (trans)rectal), intravesical,intrapulmonary, intraduodenal, intragastrical, intrathecal,subcutaneous, intramuscular, intradermal, intra-arterial, intravenous,intrabronchial, inhalation, and topical administration.

Suitable compositions and dosage forms include, for example, tablets,capsules, caplets, pills, gel caps, troches, dispersions, suspensions,solutions, syrups, granules, beads, transdermal patches, gels, powders,pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs,suppositories, liquid sprays for nasal or oral administration, drypowder or aerosolized formulations for inhalation, compositions andformulations for intravesical administration and the like. It should beunderstood that the formulations and compositions that would be usefulin the present invention are not limited to the particular formulationsand compositions that are described herein.

Oral Administration

For oral application, particularly suitable are tablets, dragees,liquids, drops, suppositories, or capsules, caplets and gelcaps. Thecompositions intended for oral use may be prepared according to anymethod known in the art and such compositions may contain one or moreagents selected from the group consisting of inert, non-toxicpharmaceutically excipients that are suitable for the manufacture oftablets. Such excipients include, for example an inert diluent such aslactose; granulating and disintegrating agents such as cornstarch;binding agents such as starch; and lubricating agents such as magnesiumstearate. The tablets may be uncoated or they may be coated by knowntechniques for elegance or to delay the release of the activeingredients. Formulations for oral use may also be presented as hardgelatin capsules wherein the active ingredient is mixed with an inertdiluent.

For oral administration, the compounds of the invention may be in theform of tablets or capsules prepared by conventional means withpharmaceutically acceptable excipients such as binding agents (e.g.,polyvinylpyrrolidone, hydroxypropylcellulose orhydroxypropylmethylcellulose); fillers (e.g., cornstarch, lactose,microcrystalline cellulose or calcium phosphate); lubricants (e.g.,magnesium stearate, talc, or silica); disintegrates (e.g., sodium starchglycollate); or wetting agents (e.g., sodium lauryl sulphate). Ifdesired, the tablets may be coated using suitable methods and coatingmaterials such as OPADRY™ film coating systems available from Colorcon,West Point, Pa. (e.g., OPADRY™ OY Type, OYC Type, Organic Enteric OY—PType, Aqueous Enteric OY-A Type, OY-PM Type and OPADRY™ White,32K18400). Liquid preparation for oral administration may be in the formof solutions, syrups or suspensions. The liquid preparations may beprepared by conventional means with pharmaceutically acceptableadditives such as suspending agents (e.g., sorbitol syrup, methylcellulose or hydrogenated edible fats); emulsifying agent (e.g.,lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily estersor ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxybenzoates or sorbic acid).

Granulating techniques are well known in the pharmaceutical art formodifying starting powders or other particulate materials of an activeingredient. The powders are typically mixed with a binder material intolarger permanent free-flowing agglomerates or granules referred to as a“granulation.” For example, solvent-using “wet” granulation processesare generally characterized in that the powders are combined with abinder material and moistened with water or an organic solvent underconditions resulting in the formation of a wet granulated mass fromwhich the solvent must then be evaporated.

Melt granulation generally consists in the use of materials that aresolid or semi-solid at room temperature (i.e. having a relatively lowsoftening or melting point range) to promote granulation of powdered orother materials, essentially in the absence of added water or otherliquid solvents. The low melting solids, when heated to a temperature inthe melting point range, liquefy to act as a binder or granulatingmedium. The liquefied solid spreads itself over the surface of powderedmaterials with which it is contacted, and on cooling, forms a solidgranulated mass in which the initial materials are bound together. Theresulting melt granulation may then be provided to a tablet press or beencapsulated for preparing the oral dosage form. Melt granulationimproves the dissolution rate and bioavailability of an active (i.e.drug) by forming a solid dispersion or solid solution.

U.S. Pat. No. 5,169,645 discloses directly compressible wax-containinggranules having improved flow properties. The granules are obtained whenwaxes are admixed in the melt with certain flow improving additives,followed by cooling and granulation of the admixture. In certainembodiments, only the wax itself melts in the melt combination of thewax(es) and additives(s), and in other cases both the wax(es) and theadditives(s) melt.

The present invention also includes a multi-layer tablet comprising alayer providing for the delayed release of one or more compounds of theinvention, and a further layer providing for the immediate release of amedication for treatment of Parkinson's Disease. Using awax/pH-sensitive polymer mix, a gastric insoluble composition may beobtained in which the active ingredient is entrapped, ensuring itsdelayed release.

Parenteral Administration

For parenteral administration, the compounds of the invention may beformulated for injection or infusion, for example, intravenous,intramuscular or subcutaneous injection or infusion, or foradministration in a bolus dose and/or continuous infusion. Suspensions,solutions or emulsions in an oily or aqueous vehicle, optionallycontaining other formulatory agents such as suspending, stabilizingand/or dispersing agents may be used.

Additional Administration Forms

Additional dosage forms of this invention include dosage forms asdescribed in U.S. Pat. Nos. 6,340,475; 6,488,962; 6,451,808; 5,972,389;5,582,837; and 5,007,790. Additional dosage forms of this invention alsoinclude dosage forms as described in U.S. Patent Applications Nos.20030147952; 20030104062; 20030104053; 20030044466; 20030039688; and20020051820. Additional dosage forms of this invention also includedosage forms as described in PCT Applications Nos. WO 03/35041; WO03/35040; WO 03/35029; WO 03/35177; WO 03/35039; WO 02/96404; WO02/32416; WO 01/97783; WO 01/56544; WO 01/32217; WO 98/55107; WO98/11879; WO 97/47285; WO 93/18755; and WO 90/11757.

Controlled Release Formulations and Drug Delivery Systems

In one embodiment, the formulations of the present invention may be, butare not limited to, short-term, rapid-offset, as well as controlled, forexample, sustained release, delayed release and pulsatile releaseformulations.

The term sustained release is used in its conventional sense to refer toa drug formulation that provides for gradual release of a drug over anextended period of time, and that may, although not necessarily, resultin substantially constant blood levels of a drug over an extended timeperiod. The period of time may be as long as a month or more and shouldbe a release which is longer that the same amount of agent administeredin bolus form.

For sustained release, the compounds may be formulated with a suitablepolymer or hydrophobic material which provides sustained releaseproperties to the compounds. As such, the compounds for use the methodof the invention may be administered in the form of microparticles, forexample, by injection or in the form of wafers or discs by implantation.

In one embodiment of the invention, the compounds of the invention areadministered to a patient, alone or in combination with anotherpharmaceutical agent, using a sustained release formulation.

The term delayed release is used herein in its conventional sense torefer to a drug formulation that provides for an initial release of thedrug after some delay following drug administration and that mat,although not necessarily, includes a delay of from about 10 minutes upto about 12 hours.

The term pulsatile release is used herein in its conventional sense torefer to a drug formulation that provides release of the drug in such away as to produce pulsed plasma profiles of the drug after drugadministration.

The term immediate release is used in its conventional sense to refer toa drug formulation that provides for release of the drug immediatelyafter drug administration.

As used herein, short-term refers to any period of time up to andincluding about 8 hours, about 7 hours, about 6 hours, about 5 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40minutes, about 20 minutes, or about 10 minutes and any or all whole orpartial increments thereof after drug administration after drugadministration.

As used herein, rapid-offset refers to any period of time up to andincluding about 8 hours, about 7 hours, about 6 hours, about 5 hours,about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40minutes, about 20 minutes, or about 10 minutes, and any and all whole orpartial increments thereof after drug administration.

Dosing

The therapeutically effective amount or dose of a compound of thepresent invention depends on the age, sex and weight of the patient, thecurrent medical condition of the patient and the progression ofSigma-receptor related disorders or diseases in the patient beingtreated. The skilled artisan is able to determine appropriate dosagesdepending on these and other factors.

A suitable dose of a compound of the present invention may be in therange of from about 0.01 mg to about 5,000 mg per day, such as fromabout 0.1 mg to about 1,000 mg, for example, from about 1 mg to about500 mg, such as about 5 mg to about 250 mg per day. The dose may beadministered in a single dosage or in multiple dosages, for example from1 to 4 or more times per day. When multiple dosages are used, the amountof each dosage may be the same or different. For example, a dose of 1 mgper day may be administered as two 0.5 mg doses, with about a 12-hourinterval between doses.

It is understood that the amount of compound dosed per day may beadministered, in non-limiting examples, every day, every other day,every 2 days, every 3 days, every 4 days, or every 5 days. For example,with every other day administration, a 5 mg per day dose may beinitiated on Monday with a first subsequent 5 mg per day doseadministered on Wednesday, a second subsequent 5 mg per day doseadministered on Friday, and so on.

In the case wherein the patient's status does improve, upon the doctor'sdiscretion the administration of the inhibitor of the invention isoptionally given continuously; alternatively, the dose of drug beingadministered is temporarily reduced or temporarily suspended for acertain length of time (i.e., a “drug holiday”). The length of the drugholiday optionally varies between 2 days and 1 year, including by way ofexample only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days,12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days,120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days,320 days, 350 days, or 365 days. The dose reduction during a drugholiday includes from 10%-100%, including, by way of example only, 10%,15%,20%,25%,30%, 35%,40%,45%,50%,55%,60%,65%,70%,75%,80%,85%,90%, 95%,or 100%.

Once improvement of the patient's conditions has occurred, a maintenancedose is administered if necessary. Subsequently, the dosage or thefrequency of administration, or both, is reduced, as a function of theviral load, to a level at which the improved disease is retained. In oneembodiment, patients require intermittent treatment on a long-term basisupon any recurrence of symptoms and/or infection.

The compounds for use in the method of the invention may be formulatedin unit dosage form. The term “unit dosage form” refers to physicallydiscrete units suitable as unitary dosage for patients undergoingtreatment, with each unit containing a predetermined quantity of activematerial calculated to produce the desired therapeutic effect,optionally in association with a suitable pharmaceutical carrier. Theunit dosage form may be for a single daily dose or one of multiple dailydoses (e.g., about 1 to 4 or more times per day). When multiple dailydoses are used, the unit dosage form may be the same or different foreach dose.

Toxicity and therapeutic efficacy of such therapeutic regimens areoptionally determined in cell cultures or experimental animals,including, but not limited to, the determination of the LD₅₀ (the doselethal to 50% of the population) and the ED₅₀ (the dose therapeuticallyeffective in 50% of the population). The dose ratio between the toxicand therapeutic effects is the therapeutic index, which is expressed asthe ratio between LD₅₀ and ED₅₀. Capsid assembly inhibitors exhibitinghigh therapeutic indices are preferred. The data obtained from cellculture assays and animal studies are optionally used in formulating arange of dosage for use in human. The dosage of such capsid assemblyinhibitors lies preferably within a range of circulating concentrationsthat include the ED₅₀ with minimal toxicity. The dosage optionallyvaries within this range depending upon the dosage form employed and theroute of administration utilized.

Those skilled in the art recognizes, or is able to ascertain using nomore than routine experimentation, numerous equivalents to the specificprocedures, embodiments, claims, and examples described herein. Suchequivalents were considered to be within the scope of this invention andcovered by the claims appended hereto. For example, it should beunderstood, that modifications in reaction conditions, including but notlimited to reaction times, reaction size/volume, and experimentalreagents, such as solvents, catalysts, pressures, atmosphericconditions, e.g., nitrogen atmosphere, and reducing/oxidizing agents,with art-recognized alternatives and using no more than routineexperimentation, are within the scope of the present application.

It is to be understood that wherever values and ranges are providedherein, all values and ranges encompassed by these values and ranges,are meant to be encompassed within the scope of the present invention.Moreover, all values that fall within these ranges, as well as the upperor lower limits of a range of values, are also contemplated by thepresent application.

The following examples further illustrate aspects of the presentinvention. However, they are in no way a limitation of the teachings ordisclosure of the present invention as set forth herein.

EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out the preferred embodiments ofthe present invention, and are not to be construed as limiting in anyway the remainder of the disclosure.

Materials and Methods Chemicals.

IPAG, haloperidol hydrochloride, rimcazole dihydrochloride, PB28dihydrochloride, BD1047, BD1063, NE100, PRE-084 hydrochloride,(+)-SKF10047 hydrochloride, and (+)-pentazocine were obtained fromTocris (Minneapolis, Minn.). (+)-Pentazocine was obtained from theNational Institute on Drug Abuse (Bethesda, Md.). The cell membranepermeable calpain and cathepsin inhibitor E64d was purchased from SigmaAldrich (St. Louis, Mo.).

Cell Lines and Transfections.

The cell lines evaluated and/or mentioned herein include: MDA-MB-468,MDA-MB-231, MCF-7, T47D, SKBR3, 4T1, PC3, DU145, LNCaP, Panc1, HepG2,HCT116, BE2C, SH-SY5Y, K562, HEK293T, and NIH3T3. All cell lines arefrom ATCC. Cells were maintained in a 1:1 mixture of DMEM:F-12 with 4.5g/liter glucose, 5% FCS, non-essential amino acids andpenicillin/streptomycin. Cells were seeded approximately 24 hours priorto start of drug treatment in most assays.

Human beclin1, human ATG5, human p97/VCP, human Sigma1, human IRE1α,human ATF4, and control siRNA were purchased from Santa CruzBiotechnology. siRNA transfections (10 nmoles per well) were performedwith INTERFERin (PolyPlus) or oligofectamine according to manufacturer'sprocedures (InVitrogen).

Cell Death Assays.

Cell death was evaluated by trypan blue exclusion assay, as well ascleaved caspase 3 (Asp 175) and cleaved PARP (Asp 214) immunoblot.Trypan blue exclusion and propidium iodide staining were used toquantify general cell death and the presence of apoptotic cell death wasconfirmed by immunoblot. The percentage of dead cells in a givenpopulation was determined by quantifying the number of trypan bluepositive (dead) cells and dividing by the total number of trypan bluepositive and negative cells.

Immunoblots and Antibodies.

Cells were lysed and proteins extracted in a modified RIPA buffer (25 mMTris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate and 0.1%SDS) supplemented with 10% glycerol (volume/volume), complete proteaseinhibitor cocktail (Roche), and Halt phosphatase inhibitor cocktail(Pierce). Approximately 10-20 μg of detergent soluble protein wereresolved on NOVEX 10-20% polyacrylamide Tris-glycine gels (InVitrogen).Immunoblots were performed in a 20 mM Tris-buffered 137 mM salinesolution (pH 7.6) containing 0.1% Tween-20 (polyoxyethylene (20)sorbitan monolaurate) and 5% (weight/volume) blotting grade non-fat drymilk (BioRad). The Lumigen PS-3 enhanced chemiluminescence kit (GEHealthcare) was used to reveal immunoblotted proteins.

The mouse anti-GFP, mouse β-actin, and rabbit Beclin1, mouse ATF4, andall horseradish peroxidase conjugated secondary antibodies werepurchased from Santa Cruz Biotechnologies. The rabbit polyclonal LC3,phospo-p38MAPK (Thr180/Tyr182), phospho-SAPK/JNK (Thr183/Tyr185), IRE1α,phospho-eIF2α (Ser51), GRP78/BiP, cleaved Caspase 3 (Asp 175), andcleaved PARP (Asp 214) were all purchased from Cell SignalingTechnologies.

Microscopy and Quantitation of Autophagosome Formation.

The human GFP-LC3 expression plasmid, pEGFP-LC3 (a gift from Drs. GraziaAmbrosini and Gary K. Schwartz, MSKCC), was stably transfected intoMDA-MB-468 and selected with 0.5 mg/ml G418 sulfate. Stable populationswere generated and compared to parental MDA-MB-468 for Sigma1 expressionand autophagic and growth inhibitory response to Sigma ligands. GFP-LC3translocation (punctae formation) was assessed by microscopy inMDA-MB-468(GFP-LC3) stable cell populations. For microscopy-basedexperiments, cells were seeded onto Lab-Tek II glass chamber slides(Nalge Nunc International). Following 24 hours of drug treatment, cellswere washed with room temperature Dulbecco modified phosphate bufferedsaline solution, containing calcium and magnesium, and fixed andpermeabilized with room temperature Cytofix-Cytoperm solution (BDBiosciences). Images of GFP-LC3 punctae were acquired with a ZeissAxioplan 2 Imaging widefield microscope using Axiovision LE software.Punctae were counted using the spot quantitation program in theFluoro-Chem software package (Alpha Innotech) and confirmed in parallelby manual counting. Autophagosome formation in MDAMB-468(GFP-LC3) cellswas quantitated as the mean number of GFP-LC3 punctae per GFP positivecell.

Autophagic Flux Assays

Autophagic flux (turnover of autolysosome cargo) was evaluated using twopreviously described methods. Lipid conjugated GFP-LC3 translocates toautophagosomes that conditionally fuse with lysosomes, leading toautolysosomal degradation of LC3 and release of GFP in the case ofactive autophagic flux. In this GFP-LC3 degradation assay, cleaved GFPwas detected by immunoblot. Autophagic flux was also verified byinhibiting autolysosomal degradation with the cell permeable calpain andcathepsin inhibitor E64d. In this assay, accumulation of LC3II was anindicator of autophagic flux

Statistical Analysis.

Statistical significance was determined by one-way ANOVA followed byBonferroni's post-test using Prism software (GraphPad).

Example 1 Induction of Dose-Responsive Activation of Autophagy

MDA-MB-468 and T47D breast adenocarcinoma cells, which natively expressSigma1, were treated with Sigma receptor antagonists or agonists. In allexperiments the antagonists, but not agonists, decreased cell size by˜20% after 24 hours of treatment with 10 μM drug (FIG. 11). In view ofrecent evidence that autophagy plays a role in cell growth, this processwas evaluated to determine whether it was activated in Sigmaantagonist-treated cells.

Initially, an established immunoblot-based assay to detect microtubuleassociated protein light chain 3 (LC3) lipidation was used to test forthe activation of autophagy. In these experiments, treatment with Sigmaantagonists (IPAG, haloperidol, rimcazole, PB28), but not agonists(PRE-084, (+)-SKF10047, (+)-pentazocine), converted LC3 to LC3II, anindication of LC3 lipid conjugation and autophagosome formation (FIG.4A). These results were confirmed with a widely used microscopy basedassay to visualize and quantify the translocation of an amino-terminalgreen fluorescent protein tagged LC3 (GFPLC3) into vesicular structures,which appear as GFP-concentrated punctae characteristic of autophagosomeformation. Since transient transfections can produce spurious GFP-LC3aggregates, stable GFP-LC3 transfected populations ofMDA-MB-468(GFP-LC3) were generated. These cells were treated for 24hours with increasing concentrations of Sigma receptor antagonists andagonists, and were compared to basal and DMSO treated controls (FIGS. 4B& 4C).

Sigma antagonist-induced autophagosome formation was dose-responsive,with a range of potencies among antagonists (FIG. 4C). Following 24hours of treatment, precipitous cell death occurred with 40 μM IPAG andhaloperidol, and with 100 μM of rimcazole and PB28. The number ofpunctae per cell produced at these concentrations were set as themaximal autophagosome induction levels (maximum effect, E_(max)) incalculating EC₅₀ values. The E_(max) of all four antagonists was 30 to35 punctae per cell, with no significant difference between ligands(FIG. 4C). However, the potency (EC₅₀+S.E.M.) of Sigma antagonists IPAG(9±3 μM), haloperidol (2±2 μM), rimcazole (50±8 μM), and PB28 (52±9 μM)varied (FIG. 4C). Basal and DMSO treated cells produced 4±1 and 5±1punctae per cell, respectively (FIG. 4C). The agonists PRE-084,(+)-SKF10047, and (+)-pentazocine produced no more than 6±2, 5±1, and6±1 punctae per cell, respectively, at drug concentrations up to 100 μM(FIG. 4C). Thus Sigma receptor antagonist treatment producedautophagosomes in a dose-responsive manner, which reached saturatinglevels, and this result was consistent with receptor-mediated effects.

Example 2 Inhibition of Sigma1 Antagonist Associated Autophagy by Sigma1RNAi

To confirm that the Sigma1 antagonist treatment associated autophagy wasindeed Sigma1 mediated, siRNA was used to knockdown Sigma1 receptors inMDA-MB-468(GFP-LC3) cells, and evaluated IPAG induced autophagy (FIGS.5A-5C). Significant Sigma1 knockdown was detectable >72 hours aftertransfection of Sigma1 selective siRNA, suggesting a stable, longprotein half-life, consistent with previous reports. Knockdown levelsreached approximately 20% of basal levels (FIG. 5A).

Autophagosome formation (GFP-LC3 punctae) and autophagic degradation(GFP-LC3 cleavage) were evaluated. Knockdown of Sigma1 alone did notinduce the formation of autophagosomes in the absence of Sigma1 ligands,6±2 punctae per cell compared to 7±3 punctae per cell in control siRNAtransfected cells (FIG. 5C). Treatment with 10 μM IPAG for 20 hoursresulted in 28±4 punctae per cell in control siRNA transfected cells anda significant inhibition to 10±2 punctae per cell in Sigma1-knockdowncells (FIG. 5C).

Example 3 Induction of ER Stress and Activation of UPR

Whether Sigma antagonists immediately induce autophagy or whether it isactivated downstream of other cellular events was next examined. AsSigma1 is highly enriched in the ER, next examined was whetherantagonist treatment could induce ER stress mediated UPR. Components ofthe IRE1α-JNK1/2 and eIF2α-ATF4 branches of the UPR as well as theUPR-associated ER chaperone, GRP78/BiP, were assayed as indicators ofactivated UPR.

The stress induced mitogen activated protein kinase p38 (p38MAPK) is adownstream target of the IRE1-TRAF2 (TNF receptor-associated receptor2)-ASK1 (apoptosis signaling regulated kinase 1) signaling complex thatis activated in response to ER stress and subsequently phosphorylatesand enhances apoptosis. In addition p38MAPK has a role in the control ofbasal and starvation-induced autophagy.

All of the above-mentioned markers of ER stress were evaluated followingtreatment with increasing doses of Sigma ligands in order to compare UPRwith the dose-responsive activation of autophagy. Sigma1 antagonist,IPAG, activated the UPR in a dose-responsive manner (FIGS. 6A-6F). Incontrast, Sigma1 agonists did not activate any of these markers (datanot shown). Interestingly, the UPR to Sigma1 antagonists induced ERstress occurs at lower doses than the autophagic response (FIGS. 6A-6F).Indeed, treatment with 1 μM IPAG, a dose that does not produceautophagosomes, resulted in a salient activation of at least sevenmarkers of UPR (FIGS. 6A-6F). Whereas, the mean EC₅₀ of LC3 lipidconjugation (i.e., LC3II induction) was 7 μM, the EC₅₀ values forinduction of ATF4, IRE1α, GRP78/BiP, and phosphorylation of eIF2α(Ser51), JNK (Thr183/Tyr185), and p38MAPK (Thr180/Tyr182) were 0.5, 0.9,1.4, 2.3, 1.6, 1.7, and 0.5 μM, respectively. These mean values,generated from two independent determinations indicated that Sigma1antagonist induction of UPR occurred at 3 to 14 fold lowerconcentrations than required for autophagosome formation (FIGS. 6A-6F).

Next, whether autophagy occurs prior or subsequent to UPR was examined.Cells were treated with 10 μM IPAG for 1, 6, 12, and 24 hours (FIGS.7A-7E). Of the six ER stress and UPR markers evaluated in thisexperiment, salient induction of 5 was detected by 1 hour of treatment,and one was clearly induced between 1 to 6 hours (FIGS. 7A-7D). Incontrast, significant formation of autophagosomes, measured by GFP-LC3punctae and LC3II immunoblot, was detected between 6 to 12 hours (FIG.7E).

Example 4 Inhibition of UPR Prevents Sigma1 Antagonist-AssociatedAutophagy

The results of the dose response and time-action experiments suggestedthat ER stress-induced UPR was engaged upstream of autophagy. However,these experiments did not demonstrate that ER stress was required toactivate autophagy. To confirm that UPR precedes and is required forSigma1 antagonist-induced autophagy, UPR was inhibited by siRNA-mediatedknockdown of IRE1α or ATF4. In these experiments, 72 hours aftertransfection of siRNA, MDA-MB-468 cells were treated for 20 hours with10 μM IPAG (FIGS. 8A-8D). Knockdown of IRE1α resulted in decreasedautophagosome formation and autophagic degradation (FIGS. 8A & 8B). Thenumber of autophagosomes per cell decreased from 24±2 when treated withIPAG to 9±2 when IRE1a was knocked down (FIG. 8C). By knocking downATF4, IPAG treatment produced 5±1 autophagosomes per cell (FIG. 8C). Inaddition to knockdown experiments, a chemical inhibitor of c-JunN-terminal kinase (JNK) signaling, SP600125, was used to inhibit theIRE1a/JNK branch of the UPR. Consistent with IRE1α knockdown, theaddition of SP600125 autophagosome formation from 23±2 (IPAG alone) to7±1 (IPAG and SP600125) in IPAG treated cell cultures (FIG. 8D).Together, these data suggested that Sigma1 antagonist-induced autophagyoccurs via UPR activation.

Example 5 Sigma1 Antagonist-Induced Autophagy Required Beclin1

To confirm that GFP-positive punctae formation and degradation wereindeed products of autophagy, the effects of RNAi mediated knockdown ofBeclin1, an essential autophagy protein, were evaluated. Knockdown ofBeclin1 significantly inhibited puncta formation, decreasing the meannumber of punctae per cell from 28±3 to 6±1 in IPAG treated cells andfrom 36±4 to 17±1 in haloperidol treated cells (FIGS. 8A-8D). Sigma1antagonist induced autophagosome formation was also inhibited by3-methyladenine (3-MA), a widely used type III phosphatidylinositol3-kinase inhibitor. Addition of 3-MA (5 mM) decreased the number of IPAGinduced punctae per cell from 23±2 to 12±1 (FIG. 12A). Treatment with 5mM 3-MA alone produced 5±1 punctae per cell, not significantly differentthan the 4±2 produced in DMSO controls (FIG. 12A).

Example 6 Inhibition of Sigma1 Antagonist-Induced UPR and AutophagyAccelerated Apoptotic Cell Death

The results described above suggested that UPR and autophagy mayfunction as primary and secondary survival responses, respectively, toSigma1 antagonist-induced ER stress. The proportion of dead MDA-MB-468cells following 24 hours of treatment with IPAG (10±2%) was notsignificantly different than DMSO treated (9±1%) control cell cultures(FIGS. 10A-10F). However, following 48 hours of continuous treatment,30±2% IPAG treated cells undergo apoptotic cell death (FIGS. 10A-10F).By 72 hours >75% of IPAG-treated cells died. Consistent with thispattern, whereas control siRNA transfected cells survived 24 hours ofIPAG (9±4%), inhibition of UPR by IRE1α knockdown potentiated thecytotoxic effect of IPAG (47±8%) at the 24-hour treatment time-point(FIGS. 10C-10D). Knockdown of ATF4 also potentiated IPAG inducedapoptosis, with 30±9% dead cells per well, whereas ATF4 knockdown alonedid not significantly alter cell death rates, with 7±2% dead cells perwell. Thus, inhibition of UPR by siRNA knockdown of IRE1α or ATF4abrogated autophagosome formation (FIGS. 8A-8D) and potentiated Sigma1antagonist-mediated apoptotic cell death (FIGS. 10C-10D).

Next, the effects of inhibiting autophagy either by siRNA mediatedBeclin1 knockdown or chemical inhibition by 3-methyladenine (3-MA) wereevaluated. Whereas treatment with 10 μM IPAG for 24 hours did not inducesignificant cell death (5±3%) with no evidence of apoptosis, inhibitingautophagosome formation with 3-MA (5 mM) or by siRNA knockdown ofBeclin1 resulted in cell death at 24 hours of IPAG treatment, with over33% dead cells per well (FIGS. 10E-10F). Whereas the percentage of deadcells per well did not significantly differ between 24 hour treatmentwith IPAG alone (5±2%) or 3-MA alone (6±2%), combined treatment withIPAG and 3-MA potentiated the number of dead cells per well to 24±5%(FIG. 12B).

Example 7

Sigma1 Receptors Associate with Proteins Involved in ER ProteinProcessing, Cell Growth, and Survival

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques andco-immunoprecipitation experiments were performed to identify andconfirm Sigma1 associated cellular factors. A plasmid constructcontaining a dual carboxy-terminal affinity-tagged Sigma1 with tandemhemagglutinin (HA) epitope and six-histidine (His6) tag, Sigma1-HA-His6was generated. This dual-tagged Sigma1 construct enabled successivehighly selective protein purification procedures. Using this approach, aSigma1-HA-His6 receptor complexes from a range of tumor cell lines,including prostate adenocarcinoma (PC3, DU145), breast adenocarcinoma(MDA-MB-468, MCF-7), neuroblastoma (BE(2)-C) was isolated. Silverstaining revealed a number of proteins that co-purified with Sigma1(FIGS. 13A-13B). LC-MS/MS analysis of the complex has identifiedapproximately 80 Sigma1-associated proteins. Preliminary data revealedthat ˜85% of Sigma1-associated proteins are directly associated with ERhomeostasis and stress response. Among these associated proteins wereGRP78/BiP and GRP94, and at least 12 heat shock family chaperones.

Example 8 Sigma Receptor Antagonist Treatment Induces ER Stress andActivation of the Unfolded Protein Response

Consistent with Sigma1 association with ER homeostatic factors,preliminary data with three prostate cancer cell lines (PC3, DU145,LaPC4) revealed an increased level of ubiquitinated proteins with Sigmaantagonist treatment, reminiscent of ERAD mediated increase inubiquitylation (data for haloperidol treatment of PC3 shown in FIGS.4A-4I). This might be due to an accumulation of ubiquitylated proteinsor due to an increase in ubiquitin ligase activity. Progression of theER stress response could be monitored by a set of markers, many of whichhave been directly linked to the UPR. The most extensively investigatedsensors that initiate the UPR are IRE1α, PERK, and ATF6, which transducesignals to a cascade of effectors. Many of these UPR 4 effectorsfunction as transcription factors that induce the synthesis of ERchaperones, such as GRP78/BiP and GRP94, involved in maintaining proteinhomeostasis. Preliminary experiments have found that treatment of PC3and DU145 PCa cells with Sigma1 antagonists, IPAG and haloperidol,resulted in salient induction of IRE1α and BiP levels (data shown forhaloperidol in FIG. 15).

Example 9 Autophagosome Formation and Autophagic Degradation

Sigma antagonist-induced ER stress led to the activation of autophagy inseveral tumor cell lines, including PC3 prostate cancer cells (FIGS.16A-16B). In preliminary experiments, an immunoblot assay was performedto detect the formation of the lipid-conjugated form of microtubuleassociated protein light chain 3 (LC3II), a widely used marker ofautophagosome formation (FIGS. 16A-16B). In addition, a well-establishedand widely used microscopy-based assay was performed which detects thetranslocation of an amino-terminal green fluorescent protein tagged LC3(GFP-LC3) into vesicular structures which appear as GFP concentratedpunctae characteristic of autophagosome formation, as shown withhaloperidol in FIGS. 6A-6F.

Sigma antagonists may induce ER stress, which in turn, leads toautophagy through a series of steps comprising the progressive stages ofUPR. The apoptosis observed with Sigma antagonist treatment is likelydue to ER stress. It is believed that autophagy functions to restore thestressed cell to homeostasis by degrading toxic proteins and damagedorganelles. However, as the cytoprotective capacity of autophagy isexceeded, the Sigma antagonist treated cell may proceed to apoptosis.However, recent work with breast adenocarcinoma supports the hypothesisthat autophagy functions as a survival response, as blockade ofautophagy markedly increases Sigma antagonist-induced apoptotic death.This multi-tiered survival response might be best described by theschematic in FIGS. 27A-27C. This may apply to prostate cancer cell linesas well; preliminary data suggest that at least some prostate cancercells may respond in a similar manner to Sigma antagonist treatment.

Example 10

Sigma1 Receptors Associate with Proteins Involved in ER ProteinProcessing, Cell Growth, and Survival

Little is known regarding the cellular role of Sigma1, its relevance toER function and thus induction of ER stress response by Sigmaantagonists. A better understanding of its mechanisms may be achieved byidentifying the proteins with which it associates. To address thisquestion, liquid chromatography-tandem mass spectrometry (LC-MS/MS)techniques and co-immunoprecipitation experiments are performed toidentify and confirm Sigma1 associated cellular factors. A plasmidconstruct is generated containing a dual carboxy-terminalaffinity-tagged Sigma1 with tandem hemagglutinin (HA) epitope andsix-histidine (His6) tag, Sigma1-HA-His6. This dual-tagged Sigma1construct permits successive highly selective protein purificationprocedures.

Using this approach, a Sigma1-HA-His6 receptor complexes is isolatedfrom a range of tumor cell lines, including prostate adenocarcinoma(PC3, DU145), breast adenocarcinoma (MDA-MB-468, MCF-7), neuroblastoma(BE(2)-C). Silver staining reveals a number of proteins that co-purifiedwith Sigma1 (FIGS. 13A-13B). LC-MS/MS analysis of the complex identifiesapproximately 80 Sigma1-associated proteins. Preliminary data revealthat ˜85% of Sigma1-associated proteins are directly associated with ERhomeostasis and stress response. Among these associated proteins areGRP78/BiP and GRP94, and at least 12 heat shock family chaperones.

Example 11

Sigma Receptor Antagonist Treatment induces ER Stress and Activation ofthe Unfolded Protein Response

As Sigma1 is highly enriched in the endoplasmic reticulum, and in lightof LC-MS/MS results, whether Sigma antagonist treatment could induce ERstress response is examined. Consistent with Sigma1 association with ERhomeostatic factors, preliminary data with three prostate cancer celllines (PC3, DU145, LaPC4) reveals an increased level of ubiquitinatedproteins with Sigma antagonist treatment, reminiscent of ERAD mediatedincrease in ubiquitylation (data for haloperidol treatment of PC3 shownin FIG. 14). Whether this is due to an accumulation of ubiquitylatedproteins or due to an increase in ubiquitin ligase activity can bedetermined.

Progression of the ER stress response can be monitored by a set ofmarkers, many of which have been directly linked to the UPR. The UPRcomprises several signaling pathways that increase the protein foldingand processing capacity of the ER in response to ER stress. The mostextensively investigated sensors that initiate the UPR, IRE1α, PERK, andATF6 transduce signals to a cascade of effectors (Marciniak et al. 2006,Cell 134:769-781; Ron et al. 2007, Nat. Rev. Mol. Cell Biol. 8:519-529;Xu et al., 2005, J. Clin. Invest 115:2656-2664; Schroder et al., 2005,Annu. Rev. Biochem. 74:739-789). Many of these UPR effectors function astranscription factors that induce the synthesis of ER chaperones, suchas GRP78/BiP and GRP94, involved in maintaining protein homeostasis(Marciniak et al. 2006, Cell 134:769-781; Ron et al. 2007, Nat. Rev.Mol. Cell Biol. 8:519-529; Xu et al., 2005, J. Clin. Invest115:2656-2664; Schroder et al., 2005, Annu. Rev. Biochem. 74:739-789).Preliminary experiments found that treatment of PC3 and DU145 PCa cellswith Sigma1 antagonists, IPAG and haloperidol, resulted in salientinduction of IRE1α and BiP levels (data shown for haloperidol in FIG.15).

Example 12 Prostate Cancer Cell Lines

By identifying the cytoprotective signaling pathways mounted in responseto Sigma antagonist treatment, more effective Sigma antagonist basedcombinations that induce ER stress and selectively block the survivalresponse are designed (FIGS. 2A-2B). This hypothesis is tested using amixed set of widely studied androgensensitive and -insensitive prostatecancer cell lines, including DU145, PC3, LaPC4, LNCaP, MDA-PCa-2a and-2b, and cell lines from the CWR22 series. Preliminary results withDU145 and PC3 in addition to published results with other Sigma receptorligand treatment of LNCaP cell lines are sensitive to Sigmaantagonist-mediated proliferation arrest and cell death (Berthois, etal., 2003, Br. J. Cancer 88:438-446; Spruce et al., 2004, Cancer Res.64:4875-4886, FIGS. 1A-1B). MDA-PCa-2a and -2b, and selected CWR22 linesare tested; these cell lines can express readily detectable levels ofprostate specific antigen and may be particularly useful in evaluatingandrogen-sensitive tumor growth and Sigma ligand response in xenograftexperiments. This mixed set of cell lines provides clues to whetherandrogen-sensitive and -insensitive prostate cancer lines respondsimilarly to Sigma antagonist-treatment.

DU145, PC3, and LNCaP cell lines have been described to express theSigma1 (Berthois, et al., 2003, Br. J. Cancer 88:438-446; Spruce et al.,2004, Cancer Res. 64:4875-4886). Sigma1 expression in MDA-PCa-2a, -2b,and CWR22 cell lines is unknown. However, Sigma1 has been detected in abroad range of tumor cell lines. In most of these lines, the number ofSigma1 binding sites has not been quantitated. Furthermore, thecorrelation between levels of Sigma1 binding sites and sensitivity toand kinetics of Sigma antagonist induced prostate cancer cell death isunclear. Therefore, Sigma1 binding sites in prostate cancer cells isfirst quantitated by radio-ligand binding assay using[³H]-(+)-pentazocine and [³H]-haloperidol. These are reference ligandsfor pharmacological characterization of Sigma binding sites, and theyare commercially available. The binding affinity (K_(d)) and the numberof binding sites per milligram of prostate cancer cell membrane(B_(max)) are determined using a standard Sigma1 binding assay protocoldescribed elsewhere (Ryan-Moro et al., 1996, Neurochem. Res.21:1309-1314).

Example 13 Sigma Receptor Ligands

Initially, the cell stress inducing properties of prototypic Sigmaligands that have been confirmed to inhibit proliferation and inducecell death of breast adenocarcinoma, neuroblastoma, leukemia, and threeof the prostate cancer cell lines described above are characterized.This set of ligands includes: haloperidol, IPAG, rimcazole, and PB28(Spruce et al., 2004, Cancer Res. 64:4875-4886; Hayashi et al., 2008,Expert Opin. Ther. Targets 12:45-58). These compounds are found toelicit ER stress response and autophagy in breast adenocarcinoma celllines. These four Sigma receptor antagonists elicit autophagy atdifferent rates and with different potencies. They also have differentselectivity for Sigma1 versus Sigma2 subtypes (Hayashi et al., 2008,Expert Opin. Ther. Targets 12:45-58; Berardi et al., 1996, J. Med. Chem.39:4255-4260; Ferris et al., 1986, Life Sci. 38:2329-2337).Interestingly, the Sigma1 selective compounds (haloperidol and IPAG) aremore potent inducers of ER stress response and autophagy than the Sigma2selective compounds (rimcazole and PB28). This is consistent with arecent report describing the potency of novel highly Sigma1 selectiveligands derived from spipethiane, supports Sigma1 selectivity ofanti-tumor Sigma ligands (Piergentili et al., J. Med. Chem.53:1261-1269). Subsequently, a broader panel of commercially availableprototypic Sigma receptor antagonists and agonists are evaluated.

Preliminary experiments with DU-145 and PC3 cells reveal activation ofUPR and autophagy following a single time-point, 24 hour treatment, witha single dose of one Sigma ligand, 10 μM haloperidol (antagonist).Therefore, the dose-responsive induction of ER stress response(including ubiquitylation, UPR, and autophagy) and cell death isevaluated. Sigma ligand potency (EC₅₀) and efficacy, in terms of themaximal induction (E_(mx)) of ER stress response and cell death, isestablished. Subsequently, the time-action of EC₂₀, EC₅₀, and ECso dosesof a selected set of effective Sigma ligands is evaluated. Time-actionexperiments help to determine if treated cells can respond to and adaptto Sigma drug induced stress at low doses. EC₅₀ and E_(max) valuesestablished here are used in experiments to evaluate drug combinationsynergy.

Example 14 Sigma Receptor Antagonist Treatment-Associated Increase inUbiquitinated Protein Levels

Immunoblot assays are performed to evaluate the time and dose-responsiveincrease of ubiquitinated protein levels in the absence and presence ofthe small synthetic peptide proteasome inhibitor MG-132 as describedelsewhere (Korolchuk et al., 2009, Mol. Cell 33:517-527). Initialimmunoblot experiments are performed with a widely used, commerciallyavailable anti-ubiquitin antibody (clone P4D1). Further experiments areperformed to compare the rate of ubiquitylation versus UPS mediateddegradation using a green fluorescent protein-tagged ubiquitin, Ub-GFP,and comparing it to a degradation resistant mutant ubiquitin,UbG76V-GFP, using an established [³⁵S]-label pulse-chase experimentalprocedure described elsewhere (Korolchuk et al., 2009, Mol. Cell33:517-527).

These experiments clarify the dose-response and kinetics of UPSinduction as well as clarify whether Sigma antagonist treatmentincreases ubiquitylation or inhibits degradation of ubiquitylatedproteins. For the goals of this proposal, these assays are used toevaluate changes in UPS mediated degradation in response to Sigmaantagonist treatment and to gauge and control for the activity ofproteasome inhibitors when used in combination with Sigma antagonists.Established ubiquitin ligase assay protocols are used in the context ofER stress and autophagy (Korolchuk et al., 2009, Mol. Cell 33:517-527;Korolchuk et al., FEBS Lett. 584:1393-1398; Gao et al., Autophagy6:126-137).

Example 15 Sigma Receptor Antagonist Treatment Associated ER Stress andActivation of the Unfolded Protein Response

Induction of these stress response markers is evaluated by immunoblot.Commercial antibodies are available for most of them. Components of theIRE1α-JNK1/2 and eIF2α-ATF4 branches of the UPR, as well as theUPR-associated ER chaperones, GRP78/BiP (FIG. 15), GRP94, and ORP150 areassayed for as indicators of activated UPR (Marciniak et al., 2006,Physiol. Rev. 86:1133-1149; Ron et al., 2007, Nat. Rev. Mol. Cell. Biol.8:519-529; Ni et al., 2007, FEBS Lett. 581:3641-3651).

Translational arrest is another indicator of stress response to unfoldedprotein accumulation in the ER. To evaluate this response immunoblotassays are performed. Phosphorylation of 4E-BP1 and eIF4E reflecttranslational arrest. The utility is confirmed in breast adenocarcinoma,wherein a time-dependent induction of markers for ER stress, IRE1α andGRP78/BiP, is observed during treatment with the Sigma antagonist IPAG35. This is accompanied by a progressive suppression of translation.Protein translational arrest is also evaluated by [³⁵S]-protein labelpulse-chase experiments. This approach quantifies protein degradation aswell as quantify translation arrest in response to Sigma drug treatment.

In one embodiment, not all cell lines express detectable levels of allUPR and stress markers (as has been experienced with breast cancer celllines). For example, PERK phosphorylation, a hallmark of UPR induction,is undetectable in many cell lines in which several other UPR markersare clearly present by other markers. Various Sigma ligands may elicitUPR by distinct mechanisms. Furthermore, different cell lines mayrespond by activating distinct branches of the UPR or distinct stressresponse pathways. Therefore, a broad panel of markers are assessed andcell lines are used in which multiple markers of UPR induction aredetectable. siRNA studies are performed to validate and confirmSigma1-mediated activities.

Example 16

Autophagosome Formation and Autophagic Degradation Associated with SigmaAntagonist Treatment

Sigma antagonist-induced ER stress leads to the activation of autophagyin several tumor cell lines, including PC3 and DU145 prostate cancercells (FIGS. 16A-16B). The established and widely used immunoblot andmicroscopy based assays described elsewhere herein are performed tocharacterize and quantify prostate cancer cell autophagic response toSigma antagonist treatment (Klionsky et al., 2008, Autophagy 4:151-175).Differences in Sigma antagonist-induced autophagosome formation areanalyzed qualitatively and quantitatively. Stable GFP-LC3 transfectantsfrom the prostate cell lines described above are generated, as describedpreviously. To confirm that Sigma antagonist-induced punctae are indeedautophagosomes, and not spurious aggregates or vesicles, controlexperiments with siRNA mediated knockdown of essential autophagyproteins such as ATG5 and Beclin1 are performed (Klionsky et al., 2008,Autophagy 4:151-175; Kuma et al., 2007, Autophagy 3:323-328). WhetherSigma antagonists induce autolysosomal degradation of cargo proteins isdetermined by using two immunoblot-based assays to detect and quantifyLC3 degradation, as described herein and in the literature (Ron et al.,2007, Nat. Rev. Mol. Cell. Biol. 8:519-529).

Example 17 Autophagy Inhibitors and Sigma Receptor Antagonists

Sigma antagonists activate autophagy in PC3 and DU145 prostate cancercells (shown for PC3 in FIGS. 16A-16B). Inhibition of autophagosomeformation or autophagic degradation by RNAi mediated knockdown ofessential autophagy components or small molecule inhibition of autophagyusing 3-methyladenine results in a salient acceleration and potentiationof Sigma antagonist-mediated apoptosis. Although useful as anexperimental tool, the clinical utility of 3-methyladenine isquestionable as it does not have good drug-like properties (Huyer etal., 2004, J. Biol. Chem. 279:38369-38378).

In addition to established autophagy inhibitors such as HCQ,combinations of Sigma antagonists with paclitaxel and vincristine, twowidely used chemotherapeutics that have been recently shown to inhibitautophagy (Groth-Pedersen et al., 2007, Cancer Res. 67:2217-2225), areexamined. Docetaxel/Sigma antagonist combinations are also examined. Invitro assays are performed including these autophagy inhibitors with theset of prostate tumor cell lines described above. Cell proliferation anddeath are evaluated as described below. In vivo, tumor xenograftexperiments are performed according to the protocol described below,with docetaxel, paclitaxel and vincristine and hydroxychloroquine dosesdescribed elsewhere (Amaravadi et al., 2007, J. Clin. Invest.117:326-336; Amaravadi et al., 2007, Clin. Cancer Res. 13:7271-7279;Groth-Pedersen et al., 2007, Cancer Res. 67:2217-2225; Canfield et al.,2006, Mol. Cancer Ther. 5:2043-2050; Kim et al., 2009, Autophagy5:567-568).

Example 18 Proteasome Inhibitors and Sigma Receptor Antagonists

Sigma antagonist-treated cells present increased levels of ubiquitinatedproteins (FIG. 14). This effect is likely due to either an increase inubiquitin ligase activity or to inhibition of proteasomal degradation(see above). Studies demonstrating the efficacy of ER stress inducingagents combined with proteasome inhibitors such as bortezomib suggestpotential for Sigma antagonist drug combinations. Sigma receptorantagonists are evaluated in combination with bortezomib and MG-132 (26Sproteasome inhibitor and calpain inhibitor) in vitro. This is extendedto prostate tumor xenograft experiments (described below).

Example 19 Molecular Chaperone Inhibitors and Sigma Receptor Antagonists

Preliminary data reveal that Sigma1 receptors bind to other molecularchaperones (FIGS. 13A-13B). If Sigma1 functions in a molecular chaperonecapacity, it is likely that Sigma1 antagonists induce ER stress byaltering its physical association with partner proteins.

Antagonist-induced stress activates protein degradation pathways inprostate adenocarcinoma cell lines (FIG. 14). Furthermore, preliminaryresults reveal a direct physical association between Sigma1 and HSP90family proteins, including GRP94. There is no reported evidence of Sigmaligand interaction with androgen receptor (AR) or AR associatedsignaling pathways. In light of preliminary data revealing Sigma1binding to HSP family chaperones and other AR associated proteins,whether Sigma antagonists can modulate AR signaling by modulating ARprotein levels, possibly by altering AR association with its cognatemolecular chaperones, is also evaluated.

Example 20 Cell Death Assays

Several monolayer culture cell death assays are performed according tothe availability of resources and need for experimental precisionregarding type and magnitude of cell death. Colorimetric Alamar Blue orMTT (yellow tetrazolium salt) assays in a 96-well format are used forinitial screens of drug combinations for their ability to decrease cellnumbers. These assays are widely used, commercially available kits.However, these assays do not directly address whether decreased cellnumbers are due to cell death or proliferation arrest or a combinationof both. Therefore, selected drug treatments are followed up with trypanblue exclusion assays to confirm that decreased cell numbers in theAlamar Blue or MTT assay are indeed due to cell death. When quantitationof both cell cycle arrest and cell death is required,flow-cytometry-based propidium iodide staining assays are performed.Whether cell death is apoptotic is determined by evaluating Caspase 3(Asp175) and PARP (Asp 214) cleavage by immunoblot, flow cytometry, ormicroscopy. These are also widely used assays to determine and quantifyapoptosis.

Whenever possible, in parallel to these cell death assays, ER stressresponse and autophagy markers are evaluated. A portion of cells fortrypan blue exclusion or propidium iodide staining assays and a portionfor protein extraction for further biochemical analysis are used. Celldeath, ER stress, and autophagy from the same treatment sample aredirectly compared.

Prior to prostate cancer cell inoculation into mice, soft agar tumorgrowth assays are performed. This is an important transitionalexperiment, as anchorage-independent, three-dimensional growth ofaggregated prostate cancer cells growing in soft agar may reactdifferently to ER stress inducing agents than in attached monolayer cellculture. The most promising drug combinations assays are evaluated insubsequent prostate tumor xenografts. In all of these in vitro assays,drug synergy is confirmed by isobolographic analyses (Zhao et al., 2004,Clin. Cancer Res. 10:7994-8004) (described elsewhere under Statisticalanalysis of drug interactions).

Example 21 Mouse Tumor Xenograft Models

The in vivo component comprises two major experimental groups: In Group1 pharmacological characterization of Sigma ligands as single agentchemotherapeutics is performed; Group 2 evaluates the anti-tumorefficacy of Sigma ligands in combination with ubiquitin proteasome andautophagy inhibiting agents.

Both normal and castrated male mice are used to compare androgensensitive versus insensitive growth of the androgen dependent andindependent prostate cell lines described above. In initial experiments,subcutaneous injection of prostate tumors are performed as described(Spruce et al., 2004, Cancer Res. 64:4875-4886; Sirotnak et al., 2002,Clin. Cancer Res. 8:3870-3876). However, as subcutaneous inoculationswould not evaluate the influence of the prostate tumor microenvironment,intraprostatic injection of prostate cancer cells is also performed(Spruce et al., 2004, Cancer Res. 64:4875-4886; Sato et al., 1997,Cancer Res. 57:1584-1589; Moussavi et al., Cancer Res. 70:1367-1376). Inthese experiments, tumor growth is tracked by measuring prostatespecific antigen levels, in the case of MDAPCa-2a, -2b, LNCaP, andselected CWR22 cell lines (Navone, et al. 2000, Clin. Cancer Res.6:1190-1197; Navone, et al. 1997, Clin. Cancer Res. 3:2493-2500; Fox etal., 2002, Clin. Cancer Res. 8:3226-3231; Agus et al., 1999, Cancer Res.59:4761-4764; Denmeade et al., 2003, Prostate 54:249-257).

Group 1: The pharmacological characterization of Sigma antagonists assingle agent anti-tumor chemotherapeutics require a number ofstandardized approaches, described below. The studies primarily evaluatetumor regression or inhibition of growth in response to treatment withSigma receptor drugs. The drugs are administered via intraperitoneal orintravenous injection.

Drug Potency and Efficacy

Potency is defined by the dose needed to produce half the maximalresponse (ED₅₀) while efficacy is defined functionally within an assayas the maximal effect (E_(max)) achieved. These are determined byexamining increasing doses of drug and measuring dose-dependent tumorregression or inhibition of tumor growth as the experimental end-points.Groups of 3-5 mice and traditional dose-responses to determine the ED₅₀and E_(max) are used. Due to the variability associated with theinoculation of cancer cells and subsequent treatment, it is essential tohave sufficient numbers of animals per set for proper statisticalevaluation. Furthermore, dose-response curves require sufficient numbersof animals per drug concentration for the assessment of the response aswell as adequate numbers of drug concentrations to define the curves andgenerate accurate ED₅₀ values and confidence limits. A typicalexperiment contains 4 drug concentrations and based upon priorutilization at least 3 experiments are anticipated to ensurereproducibility and to achieve statistically significant ED₅₀ valueswith narrow confidence limits. At least 4 of the Sigma antagonists to beevaluated are confirmed to be effective inhibitors of tumor growth inxenograft models.

Statistical Analysis of Single Agent Treatment

The statistical analyses used depend upon the type of measurement beingmade. Single comparisons are performed using either Student's t-Test,the Fisher Exact Test, or the Mann-Whitney U Test, depending on thedata. Multiple comparisons require analysis of variance (ANOVA),followed by the appropriate post-hoc analysis.

Drug Reversibility

In order to ultimately design and develop a clinical treatment protocol,it is important to determine whether the pharmacological effects ofSigma antagonists are reversible or irreversible. The reversibility ofpotential side-effects is a particularly important consideration.Therefore, a set of experiments are also performed in which drugtreatment is ceased when tumor growth is stabilized, and tumor growth inthese mice is evaluated in the same manner as in mice undergoingcontinuous drug treatment.

Group 2: In this group Sigma antagonists in combination with ubiquitinproteasome and autophagy inhibiting small molecule compounds areevaluated.

Selection of Drug Combinations for Prostate Tumor Xenograft Experiments

In vitro results guide the selection of drug combinations to be testedin vivo. In initial experiments, combinations that include Sigmaantagonists with HCQ (autophagy inhibitor) or bortezomib (proteasomeinhibitor) are used. Bortezomib and HCQ doses and treatment intervalsare guided by published protocols (Williams et al., 2003, Mol. Cancer.Ther. 2:835-843: Williams et al., 2003, Cancer Res. 63:7338-7344;Amaravadi et al., 2007, J. Clin. Invest. 117:326-336; Amaravadi et al.,2011, Clin. Cancer Res. 17:654-666). Sigma antagonist doses are based onthe dose-response studies performed above. Interactions among drugs andtargets within tumors offer opportunities to optimize efficacy withoutincreasing side-effects. Thus, although efficacy in tumor growthinhibition or regression is the primary read-out, potential side-effectsof Sigma antagonists alone and in drug combinations are monitored.

Statistical Analysis of Drug Interactions

Drug interactions are evaluated using isobolographic analysis. Theprimary goal in these types of studies is to determine whether or notthe drug interactions/combinations demonstrate synergy or simpleadditive effects. In this approach, the ED₅₀ for each drug or each siteis determined and their ratio established. Dose-response curves for theinteractions using this ratio are performed and the ED₅₀ for thecombination is determined. The results are then plotted. The ED₅₀ valuefor each individual drug is plotted on either the X- or Y-axis. The ED₅₀for the combination is added to the plot. If it lies on a lineconnecting the two individual determinations, the result is additive. Ifit falls below the line, the interactions are synergistic. If it fallsabove the line, they are antagonistic. The dose-response curves areperformed as described above.

Biochemical Analysis of Xenografted Prostate Tumors

At the end of each treatment course, tumors and organs are resected,including liver and whole brain, postmortem. Tumors are analyzed forevidence ofUPR, ER stress response, ubiquitylation, cell proliferation,autophagy, and apoptosis. Organs are obtained for biochemical studies inorder to evaluate the effects of Sigma antagonist and drug combinationtreatment on other tissues, in order to help predict side-effects suchas hepatotoxicity and potential neurotoxicity. The harvested prostatetumor is divided into three fragments for: (1) protein extraction andbiochemical analysis (e.g., immunoblots to evaluate the markers andproteins described above); (2) mRNA extraction for RT-PCR experiments(e.g., when using XBP-1 splicing as a marker for UPR or when changes intranscription or mRNA stability or turnover of selected are suspected);(3) formalin fixed for immunohistochemistry (IHC) experiments. IHCprocedures are used to evaluate Sigma1 (rabbit polyclonal and monoclonalAbs, mouse and hamster monoclonals generated in our lab), UPR markers(GRP78/BiP), autophagy (LC3II), apoptosis (cleaved Caspase 3),

As demonstrated herein, Sigma1 antagonists, but not agonists, inducedendoplasmic reticulum (ER) stress and subsequent unfolded proteinresponse (UPR) (FIGS. 4A-4I, 5A-5C, 6A-6F, 7A-7E, 8A-8D). Severe orprolonged Sigma1 antagonist-induced ER stress appeared to overwhelm thecytoprotective, adaptive capacity of the UPR and autophagy was engagedas a secondary response. Treatment with four Sigma1 antagonists (IPAG,haloperidol, rimcazole, PB28) resulted in autophagosome formation andflux. However, all three agonist did not induce UPR or autophagy. Inaddition to affinity to Sigma receptors, these compounds also bind toother receptor systems. For example, rimcazole binds to the DAT dopaminetransporter with higher affinity than the Sigma receptor system.Haloperidol binds to D2 dopamine receptors and Sigma receptors withsimilar affinity. The Sigma ligands used in this study also have beendescribed to have varying affinity and selectivity for Sigma receptorsubtypes. For example, IPAG (K_(i) 5±2 nM) has significantly higheraffinity for the Sigma1 than does rimcazole (K_(i) 80±22 nM).Interestingly, the two antagonists with greater Sigma1 binding affinity,haloperidol and IPAG, were significantly more potent inducers ofautophagy (FIGS. 4A-4I). The results of RNAi assays are consistent withSigma1 as the principal mediator of this effect (FIGS. 5A-5C).

Without wishing to be limited by theory, a possible explanation for theabsence of agonist effect may be the predominance of receptors in aconstitutive agonist conformation. Alternatively, because IPAG andhaloperidol associated autophagosome formation is not blocked by PRE084and (+)-SKF10047, it is possible that antagonists and agonists binddistinct regions of the receptor and thereby elicit different effects.

1-72. (canceled)
 73. A method of treating neuropathic pain in a subject,the method comprising administering to the subject a compound of Formula(II):R^(A)-R^(B)  (II), wherein: R^(A) is selected from the group consistingof:

X⁴ is selected from the group consisting of OCH₃, F, Cl, Br, and I; andR^(B) is selected from the group consisting of:

or a salt, solvate, or N-oxide thereof; and any combinations thereof.74. The method of claim 73, wherein the compound is selected from thegroup consisting of1-(3-(4-fluorophenoxy)propyl)-3-(4-iodophenyl)guanidine (Compound A);1-(3-(4-fluorophenoxy)propyl)-3-(4-methoxyphenyl)guanidine (Compound B);1-(3-(4-fluorophenoxy)propyl)-3-(4-chlorophenyl)guanidine (Compound G);or a salt, solvate or N-oxide thereof, and any combinations thereof. 75.The method of claim 73, wherein R^(A) is


76. The method of claim 73, wherein R^(A) is


77. The method of claim 76, wherein X⁴ is selected from the groupconsisting of F, Cl, Br, and I.
 78. The method of claim 76, wherein X⁴is OCH₃.
 79. The method of claim 73, wherein R^(A) is


80. The method of claim 73, wherein the compound is1-(3-(4-fluorophenoxy)propyl)-3-(4-iodophenyl)guanidine (Compound A), ora salt, solvate or N-oxide thereof, and any combinations thereof. 81.The method of claim 73, wherein the compound is1-(3-(4-fluorophenoxy)propyl)-3-(4-methoxyphenyl)guanidine (Compound B),or a salt, solvate or N-oxide thereof, and any combinations thereof. 82.The method of claim 73, wherein the compound is1-(3-(4-fluorophenoxy)propyl)-3-(4-chlorophenyl)guanidine (Compound G),or a salt, solvate or N-oxide thereof, and any combinations thereof. 83.The method of claim 73, wherein the compound is part of a pharmaceuticalcomposition, which is formulated for at least one selected from thegroup consisting of sustained release, delayed release, and pulsatilerelease.
 84. The method of claim 73, wherein the compound isadministered to the subject by at least one administration routeselected from the group consisting of oral, parenteral, transdermal,transmucosal, intravesical, intrapulmonary, intraduodenal,intragastrical, intrathecal, subcutaneous, intramuscular, intradermal,intra-arterial, intravenous, intrabronchial, inhalation, and topical.85. The method of claim 73, wherein the compound is part of apharmaceutical composition, which is a cream or lotion.
 86. The methodof claim 73, wherein the compound is part of a pharmaceuticalcomposition, which further comprises least one additional therapeuticagent.
 87. The method of claim 73, wherein the compound is part of apharmaceutical composition, which is administered in the form of atransdermal patch.
 88. The method of claim 74, wherein the compound ispart of a pharmaceutical composition, which is administered in the formof a transdermal patch.
 89. The method of claim 73, wherein the subjectis a mammal.
 90. The method of claim 89, wherein the mammal is a human